Xiangpeng Kong, Ph.D.

Human Anti-V3 HIV-1 Monoclonal Antibodies Encoded by the VH5-51/VL Lambda Genes Define a Conserved Antigenic Structure
Gorny, Miroslaw K; Sampson, Jared; Li, Huiguang; Jiang, Xunqing; Totrov, Maxim; Wang, Xiao-Hong; Williams, Constance; O'Neal, Timothy; Volsky, Barbara; Li, Liuzhe; Cardozo, Timothy; Nyambi, Phillipe; Zolla-Pazner, Susan; Kong, Xiang-Peng
2011 ;6(12):e27780-e27780, PLoS ONE
Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs
— id: 146267, year: 2011, vol: 6, page: e27780, stat: Journal Article,

Crystal structures of human anti-V2 mAbs 697-30D and 8. 9D and what we can learn from their antigen-binding sites
Pan R.; Sampson J.M.; Spurrier B.; Totrov M.; O'Neal T.; Williams C.; Boliar S.; Allen S.; Mulenga J.; Robinson J.; Derdeyn C.A.; Gorny M.K.; Zolla-Pazner S.; Kong X.
2011 ;27(10):A120-A121, AIDS research & human retroviruses
Background: The immunogenic V2 region of HIV-1 gp120 has been largely overlooked as a target for AIDS vaccine discovery. However, recent studies of sera from vaccinees in RV144 trial suggested that anti-V2 Abs were elicited and possibly contributed to protection. Structural understanding of human anti-V2 mAbs and their epitopes can facilitate design of immunogens. Methods: We determined crystal structures of Fab fragments of two human anti-V2 mAbs 697-30D and 8. 9D, both at a resolution of 2. 5 A, and analyzed their antigen-binding sites (ABS) and possible modes of interactions with V1V2 of gp120. Results: MAb 697-30D, from a subtype B virus infected subject and encoded by VH1-69 gene, is a broadly cross-reactive anti-V2 mAb able to neutralize Tier 1 pseudoviruses; its epitope was mapped to conserved residues in V2. Structural analysis of Fab 697-30D revealed that its ABS consists of two distinct regions: (1) A surface pocket is located at the center of CDR loops formed by large aromatic residues, and it can accommodate residues with large side chains in the epitope identified by functional studies. (2) A convex hydrophobic surface is comprised of a cluster of CDR H2/H3 residues. Comparison with structures of other VH1- 69 mAbs suggests that mAb 697-30D likely binds to the region of a short helix or a relatively flat surface of V2. Autologous neutralizing mAb 8. 9D was isolated from a subtype C infected subject, and its epitope was mapped to the stem of V1V2. Its ABS is split by the upward positioned Tyr100b of CDR H3 into a positively-charged side and a negatively-charged side. Surface pockets in these regions can bind side chains of charged residues of V1V2, facilitating escape by mutations. Conclusion: Crystal structures of human anti-V2 mAbs provide structure-function insights of their epitopes. This information may contribute to rational design of immunogens targeting V2 region of gp120
— id: 139487, year: 2011, vol: 27, page: A120, stat: Journal Article,

Structural Analysis of Human and Macaque mAbs 2909 and 2.5B: Implications for the Configuration of the Quaternary Neutralizing Epitope of HIV-1 gp120
Spurrier, Brett; Sampson, Jared M; Totrov, Maxim; Li, Huiguang; O'Neal, Timothy; Williams, Constance; Robinson, James; Gorny, Miroslaw K; Zolla-Pazner, Susan; Kong, Xiang-Peng
2011 May 11;19(5):691-699, Structure
The quaternary neutralizing epitope (QNE) of HIV-1 gp120 is preferentially expressed on the trimeric envelope spikes of intact HIV virions, and QNE-specific monoclonal antibodies (mAbs) potently neutralize HIV-1. Here, we present the crystal structures of the Fabs of human mAb 2909 and macaque mAb 2.5B. Both mAbs have long beta hairpin CDR H3 regions >20 A in length that are each situated at the center of their respective antigen-binding sites. Computational analysis showed that the paratopes include the whole CDR H3, while additional CDR residues form shallow binding pockets. Structural modeling suggests a way to understand the configuration of QNEs and the antigen-antibody interaction for QNE mAbs. Our data will be useful in designing immunogens that may elicit potent neutralizing QNE Abs
— id: 132586, year: 2011, vol: 19, page: 691, stat: Journal Article,

"Corrigendum to Structure-guided design and immunological characterization of immunogens presenting the HIV-1 gp120 V3 loop on a CTB scaffold"" [Virology 351 (2010) 513-523]"
Totrov M.; Jiang X.; Kong X.-P.; Cohen S.; Krachmarov C.; Salomon A.; Williams C.; Seaman M.S.; Abagyan R.; Cardozo T.; Gorny M.K.; Wang S.; Lu S.; Pinter A.; Zolla-Pazner S.
2011 ;409(2):360-360, Virology
— id: 119242, year: 2011, vol: 409, page: 360, stat: Journal Article,

V2-reactive antibodies in RV144 vaccinees' plasma
Zolla-Fazner S.; Cardozo T.; De Camp A.; Haynes B.; Kim J.; Kong X.; Michael N.; Rerks-Ngarm S.; Williams C.
2011 ;27(10):A21-A21, AIDS research & human retroviruses
Background: Initial studies suggested that V2 antibodies (Abs) played a protective role in RV144. Filot experiments were needed to quantify and characterize anti-V2 Abs in plasma of RV144 participants. Methods: One hundred coded RV144 pilot study specimens were analyzed by ELISA using plasma obtained before immunization (visit 1), and 2 and 28 weeks after the last immunizing dose (visits 8 and 9, respectively). Results were decoded after submission and analysis of data. Results: [a] At visit 1, 0/20 placebo and 3/80 vaccinee specimens diluted 1:20 reacted with gp120JR-FL. At visit 8, 0/20 and 80/80 were reactive. Flasma were not reactive vs. bovine serum albumin. [b] At visit 1, 0/20 placebo and 1/80 vaccinee specimens diluted 1:20 reacted with a V1V2HxB2-gp70 fusion protein. At visit 8, 1/20 and 76/80 (95%) were reactive. [c] Reactivity was tested against four 21-mer linear V2 peptides spanning the a4/?7 binding motif, chosen to represent: the most polar sequence found in V2 loops with 38 amino acids ('polar-38'); the most common sequence of V2s with 40 AAs ('common-40'); the most polar sequence in V2 loops with 40 AAs ('polar-40'); and the consensus sequence of V2 loops with 40 AAs ('consensus-40'). Activity in plasma diluted 1:100 showed differential reactivity with the peptides: 'polar-38' & 'polar-40'>'consensus'>'common- 40'. For visit 8, 1/20 placebo and 47/80 (59%) vaccine specimens were reactive with 'polar-38'. Shorter linear peptides were poorly reactive. Notably, by visit 9, the reactivity to gp120_JR-FL, V1V2_HxB2-gp70, and all V2 peptides was markedly reduced in all vaccinees' plasmas. Conclusion: Anti-V2 Abs were elicited by the RV144 immunization protocol. Reactivity was highest 2 weeks after the last boost, and was significantly diminished 26 weeks later. V2-spe- cific reactivity was demonstrable with a fusion protein containing the entire V1V2 loop and with 21-mer linear V2 peptides spanning the a4/?7 binding motif
— id: 139488, year: 2011, vol: 27, page: A21, stat: Journal Article,

Cross-Clade HIV-1 Neutralizing Antibodies Induced with V3-Scaffold Protein Immunogens following Priming with gp120 DNA
Zolla-Pazner, Susan; Kong, X-P; Jiang, Xunqing; Cardozo, Timothy; Nadas, Arthur; Cohen, Sandra; Totrov, Maxim; Seaman, Michael S; Wang, Shixia; Lu, Shan
2011 Oct;85(19):9887-9898, Journal of virology
The V3 epitope is a known target for HIV-1 neutralizing antibodies (NAbs), and V3-scaffold fusion proteins used as boosting immunogens after gp120 DNA priming were previously shown to induce NAbs in rabbits. Here, we evaluated whether the breadth and potency of the NAb response could be improved when boosted with rationally designed V3-scaffold immunogens. Rabbits were primed with codon-optimized clade C gp120 DNA and boosted with one of five V3-cholera toxin B fusion proteins (V3-CTBs) or with double combinations of these. The inserts in these immunogens were designed to display V3 epitopes shared by the majority of global HIV-1 isolates. Double combinations of V3-CTB immunogens generally induced more broad and potent NAbs than did boosts with single V3-CTB immunogens, with the most potent and broad NAbs elicited with the V3-CTB carrying the consensus V3 of clade C (V3(C)-CTB), or with double combinations of V3-CTB immunogens that included V3(C)-CTB. Neutralization of tier 1 and 2 pseudoviruses from clades AG, B, and C and of peripheral blood mononuclear cell (PBMC)-grown primary viruses from clades A, AG, and B was achieved, demonstrating that priming with gp120 DNA followed by boosts with V3-scaffold immunogens effectively elicits cross-clade NAbs. Focusing on the V3 region is a first step in designing a vaccine targeting protective epitopes, a strategy with potential advantages over the use of Env, a molecule that evolved to protect the virus by poorly inducing NAbs and by shielding the epitopes that are most critical for infectivity
— id: 137442, year: 2011, vol: 85, page: 9887, stat: Journal Article,

Map of broad and narrow neutralization in the V3 loop crown
Almond D.; Kong X.; Zolla-Pazner S.; Cardozo T.
2010 ;26(10):A27-A28, AIDS research & human retroviruses
Background: Sequence variability of the V3 loop crown has often been considered an impediment to eliciting broadly neutralization antibodies targeted to this region. Our work maps contacts between human anti-V3 monoclonal antibodies (mAbs) and the V3 crown in an attempt to make a structural distinction between the sites targeted by broadly as opposed to narrowly neutralizing mAbs. Methods: The 3D structure of the 16-residue V3 crown bound to anti-V3 mAb 2219 was divided into 4 regions (stem, hydrophilic patch, hydrophobic patch and turn) based on previously published analyses (Almond et. al., 2010). Known mAb/V3 contacts from Jiang et al., 2010 and others were then mapped onto this structure and compared to measurements of neutralization breadth by the respective mAbs in infectivity assays. Results: mAbs that entirely engage the less sequence variable zones of the V3 loop (the stem, turn and hydrophobic zones) tend to be more broadly neutralizing than mAbs that contact at least one side-chain in the most variable region of V3 (the hydrophilic zone). The contact in the highly variable region appears to narrow the Ab reactivity regardless of how many other contacts are formed with V3 backbone atoms or with side-chains in the conserved regions. Conclusion: Broadly neutralizing mAbs are targeted to the structurally conserved region, and they avoid contact with the highly sequence-variable zone. More narrowly neutralizing Abs make at least one side-chain contact within the highly-sequence variable zone: these latter mAbs are vulnerable to a single escape mutation at that side-chain/amino acid location, and indeed these side-chains are frequently mutated in circulating viruses. This knowledge could be exploited for HIV vaccine design by designing immunogens that immunologically silence the variable zone in the V3 loop
— id: 114523, year: 2010, vol: 26, page: A27, stat: Journal Article,

Structural conservation predominates over sequence variability in the crown of HIV type 1's V3 loop
Almond, David; Kimura, Tetsuya; Kong, XiangPeng; Swetnam, James; Zolla-Pazner, Susan; Cardozo, Timothy
2010 Jun;26(6):717-723, AIDS research & human retroviruses
The diversity of HIV-1 is a confounding problem for vaccine design, as the human immune response appears to favor poor or strain-specific responses to any given HIV-1 virus strain. A significant portion of this diversity is manifested as sequence variability in the loops of HIV-1's surface envelope glycoprotein. Here we show that the most variable sequence positions in the third variable (V3) loop crown cluster to a small zone on the surface of one face of the V3 loop ss-hairpin conformation. These results provide a novel visualization of the gp120 V3 loop, specifically demonstrating a surprising preponderance of conserved three-dimensional structure in a highly sequence-variable region. From a structural point of view, there appears to be less diversity in this region of the HIV-1 'principle neutralizing domain' than previously appreciated
— id: 110086, year: 2010, vol: 26, page: 717, stat: Journal Article,

Engineered immunogen presenting an epitope recognized by a neutralizing mAb elicits mammalian serum that recapitulate the mAb's specificity
Cardozo T.; Kong X.; Totrov M.; Wang S.; Lu S.; Gorny M.; Pinter A.; Seaman M.; Zolla-Pazner S.
2010 ;26(10):A26-A27, AIDS research & human retroviruses
Background: To exploit the promising properties of cross-strain neutralizing monoclonal antibodies (mAbs), immunogens should elicit polyclonal Ab responses in mammals that mimic the reactivity of these mAbs. To demonstrate the feasibility of this approach, the epitope recognized by the anti-V3 loop mAb 3074- which is present in approximately 90% of circulating HIV-1 viruses-was use as a template for immunogen design. Methods: A V3 loop-cholera toxin B fusion protein (V3₃₀₇₄-CTB) was designed to eliminate the epitopes targeted by several anti-V3 loop mAbs while preserving the epitope targeted by mAb 3074. New Zealand White rabbits were primed with codon-optimized clade C gp120 DNA and then boosted with V3₃₀₇₄-CTB. Anti-V3 mAbs and rabbit immune sera were assessed for neutralizing activity against (a) V3 chimeric psVs infecting U87 CD4 + CCR5 + cells, (b) primary isolates infecting TZM-bl cells, (c) Tier 1 and (d) Tier 2 psVs infecting TZM.bl cells. Results: V33074-CTB bound specifically to mAb 3074 but not to several other anti-V3 mAbs. A psV bearing the V3₃₀₇₄-designed sequence was neutralized only by mAb 3074. Immune sera elicited in rabbits using V3₃₀₇₄-CTB demonstrated 50% neutralization of (a) 7/7 V3 chimeric psVs carrying the V3 loops of clades A1, AG, AE, B, C, F, and H, (b) 3/10 primary isolates from clades A, AG and B, (c) 4/4 Tier 1 viruses, and (d) 4/14 Tier 2 viruses from clades B and C. Little neutralizing activity was seen in the immune sera against a V3 chimeric psV lacking the 3074 epitope, and the only three Tier 2 clade C viruses neutralized by mAb 3074 were also the only three Tier 2 Clade C viruses neutralized by the serum of the best responder. Conclusion: Our results demonstrate that V3₃₀₇₄-CTB elicited a polyclonal, cross-strain neutralizing Ab response in mammalian serum mirroring the specificity of 3074-the mAb used as a template for immunogen design
— id: 114522, year: 2010, vol: 26, page: A26, stat: Journal Article,

Germline variable genes code for contact residues maintained during affinity maturation of human anti-V3 monoclonal antibodies encoded by VH5-51
Gorny M.K.; Sampson J.; Li H.; Jiang X.; Totrov M.; Wang X.; Li L.; Williams C.; Luthra K.; Nyambi P.; Zolla-Pazner S.; Kong X.
2010 ;26(10):A25-A25, AIDS research & human retroviruses
Background: Immunogenetic studies have revealed the importance of certain immunoglobulin (Ig) genes encoding monoclonal antibodies (mAbs) against various epitopes in the envelope of HIV-1. We have demonstrated recently that VH5-51 gene segment is preferentially utilized by 18 (35%) of 51 human anti-V3 mAbs. Methods: In this study, a panel of 18 anti-V3 mAbs were examined which were derived from individuals infected with clade B and non-clade B HIV-1; all were encoded by the VH5-51 gene segment and neutralized Tier 1 and Tier 2 viruses. Immunochemical and crys-tallographic methods were used to study their function and the structure of the antigen-combining site. Results: The VH5-51 gene used by all 18 mAbs paired only with lambda light chain genes, mainly with VL1-47 and VL3-1 genes. This restricted pairing of Ig genes resulted in the formation of a conserved antigen combining site, as documented by crystallo-graphic studies of five of these anti-V3 Fabs in complex with V3 peptides. The VH5-51-encoded V3mAbs recognize slight variations of an epitope which contains conserved residues in the N- and C-terminal b-strands of the V3 crown. This finding was confirmed by the binding of the mAbs to a peptide which mimics this region of V3 but lacks the tip of the V3 loop. Crystallographic analysis of the Fab/peptide complex showed that all contact residues in the CDR domains, except CDR H3 and L3, are germline-encoded and thus determine the conserved character of the binding site. The data indicate that affinity maturation of these mAbs has preserved the germline-encoded interaction with the antigen. Conclusion: The immunogenetic studies of anti-V3 neutralizing mAbs revealed that certain germline variable genes encode the contact residues which are maintained during antibody evolution; targeting epitopes preferentially recognized by such Ig genes with vaccines should induce broadly reactive neutralizing Ab responses
— id: 114521, year: 2010, vol: 26, page: A25, stat: Journal Article,

Conserved structural elements in the V3 crown of HIV-1 gp120
Jiang, Xunqing; Burke, Valicia; Totrov, Maxim; Williams, Constance; Cardozo, Timothy; Gorny, Miroslaw K; Zolla-Pazner, Susan; Kong, Xiang-Peng
2010 Aug;17(8):955-961, Nature structural & molecular biology
Binding of the third variable region (V3) of the HIV-1 envelope glycoprotein gp120 to the cell-surface coreceptors CCR5 or CXCR4 during viral entry suggests that there are conserved structural elements in this sequence-variable region. These conserved elements could serve as epitopes to be targeted by a vaccine against HIV-1. Here we perform a systematic structural analysis of representative human anti-V3 monoclonal antibodies in complex with V3 peptides, revealing that the crown of V3 has four conserved structural elements: an arch, a band, a hydrophobic core and the peptide backbone. These are either unaffected by or are subject to minimal sequence variation. As these regions are targeted by cross-clade neutralizing human antibodies, they provide a blueprint for the design of vaccine immunogens that could elicit broadly cross-reactive protective antibodies
— id: 111542, year: 2010, vol: 17, page: 955, stat: Journal Article,

Structures of human mAb 2909 and rhesus mAb 2.5B that target quaternary structure-dependent neutralizing epitopes of HIV-1 gp120
Kong, X.; Spurrier, B.; Sampson, J.; Totrov, M.; Williams, C.; Robinson, J.; Gorny, M. K.; Zolla-Pazner, S.
2010 OCT ;26(10):A56-A57, AIDS research & human retroviruses
— id: 117317, year: 2010, vol: 26, page: A56, stat: Journal Article,

A new activity of anti-HIV and anti-tumor protein GAP31: DNA adenosine glycosidase--structural and modeling insight into its functions
Li, Hui-Guang; Huang, Philip L; Zhang, Dawei; Sun, Yongtao; Chen, Hao-Chia; Zhang, John; Huang, Paul L; Kong, Xiang-Peng; Lee-Huang, Sylvia
2010 Jan 1;391(1):340-345, Biochemical & biophysical research communications
We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA
— id: 106367, year: 2010, vol: 391, page: 340, stat: Journal Article,

Structure-guided design and immunological characterization of immunogens presenting the HIV-1 gp120 V3 loop on a CTB scaffold
Totrov, Maxim; Jiang, Xunqing; Kong, Xiang-Peng; Cohen, Sandra; Krachmarov, Chavdar; Salomon, Aidy; Williams, Constance; Seaman, Michael S; Abagyan, Ruben; Cardozo, Timothy; Gorny, Miroslaw K; Wang, Shixia; Lu, Shan; Pinter, Abraham; Zolla-Pazner, Susan
2010 Sep 30;405(2):513-523, Virology
V3 loop is a major neutralizing determinant of the HIV-1 gp120. Using 3D structures of cholera toxin B subunit (CTB), complete V3 in the gp120 context, and V3 bound to a monoclonal antibody (mAb), we designed two V3-scaffold immunogen constructs (V3-CTB). The full-length V3-CTB presenting the complete V3 in a structural context mimicking gp120 was recognized by the large majority of our panel of 24 mAbs. The short V3-CTB presenting a V3 fragment in the conformation observed in the complex with the 447-52D Fab, exhibited high-affinity binding to this mAb. The immunogens were evaluated in rabbits using DNA-prime/protein-boost protocol. Boosting with the full-length V3-CTB induced high anti-V3 titers in sera that potently neutralize multiple HIV virus strains. The short V3-CTB was ineffective. The results suggest that very narrow antigenic profile of an immunogen is associated with poor Ab response. An immunogen with broader antigenic activity elicits robust Ab response
— id: 133786, year: 2010, vol: 405, page: 513, stat: Journal Article,

Analysis of neutralizing antibody responses induced by gp120 DNA prime followed by monovalent or polyvalent V3 epitope boost
Wang, S.; Lu, S.; Kong, X.; Cardozo, T.; Cohen, S.; Jiang, X.; Totrov, M.; Pinter, A.; Krachmarov, C.; Seaman, M.; Zolla-Pazner, S.
2010 OCT ;26(10):A54-A54, AIDS research & human retroviruses
— id: 117315, year: 2010, vol: 26, page: A54, stat: Journal Article,

Sequence variability in the crown of the V3 loop of the HIV-1 envelope is clustered within a small 3D structural zone
Almond, D; Kimura, T; Kong, X; Swetnam, J; Zolla-Pazner, S; Cardozo, T
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105709, year: 2009, vol: 6, page: 115, stat: Journal Article,

Structural basis of the cross-reactivity of genetically related human anti-HIV-1 mAbs: implications for design of V3-based immunogens
Burke, Valicia; Williams, Constance; Sukumaran, Madhav; Kim, Seung-Sup; Li, Huiguang; Wang, Xiao-Hong; Gorny, Miroslaw K; Zolla-Pazner, Susan; Kong, Xiang-Peng
2009 Nov 11;17(11):1538-1546, Structure
Human monoclonal antibodies 447-52D and 537-10D, both coded by the VH3 gene and specific for the third variable region (V3) of the HIV-1 gp120, were found to share antigen-binding structural elements including an elongated CDR H3 forming main-chain interactions with the N terminus of the V3 crown. However, water-mediated hydrogen bonds and a unique cation-pi sandwich stacking allow 447-52D to be broadly reactive with V3 containing both the GPGR and GPGQ crown motifs, while the deeper binding pocket and a buried Glu in the binding site of 537-10D limit its reactivity to only V3 containing the GPGR motif. Our results suggest that the design of immunogens for anti-V3 antibodies should avoid the Arg at the V3 crown, as GPGR-containing epitopes appear to select for B cells making antibodies of narrower specificity than V3 that carry Gln at this position
— id: 105343, year: 2009, vol: 17, page: 1538, stat: Journal Article,

Mutations in the beta-tubulin gene TUBB2B result in asymmetrical polymicrogyria
Jaglin, Xavier Hubert; Poirier, Karine; Saillour, Yoann; Buhler, Emmanuelle; Tian, Guoling; Bahi-Buisson, Nadia; Fallet-Bianco, Catherine; Phan-Dinh-Tuy, Francoise; Kong, Xiang Peng; Bomont, Pascale; Castelnau-Ptakhine, Laetitia; Odent, Sylvie; Loget, Philippe; Kossorotoff, Manoelle; Snoeck, Irina; Plessis, Ghislaine; Parent, Philippe; Beldjord, Cherif; Cardoso, Carlos; Represa, Alfonso; Flint, Jonathan; Keays, David Anthony; Cowan, Nicholas Justin; Chelly, Jamel
2009 Jun;41(6):746-752, Nature genetics
Polymicrogyria is a relatively common but poorly understood defect of cortical development characterized by numerous small gyri and a thick disorganized cortical plate lacking normal lamination. Here we report de novo mutations in a beta-tubulin gene, TUBB2B, in four individuals and a 27-gestational-week fetus with bilateral asymmetrical polymicrogyria. Neuropathological examination of the fetus revealed an absence of cortical lamination associated with the presence of ectopic neuronal cells in the white matter and in the leptomeningeal spaces due to breaches in the pial basement membrane. In utero RNAi-based inactivation demonstrates that TUBB2B is required for neuronal migration. We also show that two disease-associated mutations lead to impaired formation of tubulin heterodimers. These observations, together with previous data, show that disruption of microtubule-based processes underlies a large spectrum of neuronal migration disorders that includes not only lissencephaly and pachygyria, but also polymicrogyria malformations
— id: 135247, year: 2009, vol: 41, page: 746, stat: Journal Article,

Molecular design of a mimotope that preserves conserved structural elements of the HIV-1 V3 crown
Jiang, X; Totrov, M; Sampson, J; Williams, C; Gorny, MK; Zollla-Pazner, S; Kong, X
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105702, year: 2009, vol: 6, page: 115, stat: Journal Article,

Structural and functional studies of Aspergillus oryzae cutinase: enhanced thermostability and hydrolytic activity of synthetic ester and polyester degradation
Liu, Zhiqiang; Gosser, Yuying; Baker, Peter James; Ravee, Yaniv; Lu, Ziying; Alemu, Girum; Li, Huiguang; Butterfoss, Glenn L; Kong, Xiang-Peng; Gross, Richard; Montclare, Jin Kim
2009 Nov 4;131(43):15711-15716, Journal of the American Chemical Society
Cutinases are responsible for hydrolysis of the protective cutin lipid polyester matrix in plants and thus have been exploited for hydrolysis of small molecule esters and polyesters. Here we explore the reactivity, stability, and structure of Aspergillus oryzae cutinase and compare it to the well-studied enzyme from Fusarium solani. Two critical differences are highlighted in the crystallographic analysis of the A. oryzae structure: (i) an additional disulfide bond and (ii) a topologically favored catalytic triad with a continuous and deep groove. These structural features of A. oryzae cutinase are proposed to result in an improved hydrolytic activity and altered substrate specificity profile, enhanced thermostability, and remarkable reactivity toward the degradation of the synthetic polyester polycaprolactone. The results presented here provide insight into engineering new cutinase-inspired biocatalysts with tailor-made properties
— id: 133731, year: 2009, vol: 131, page: 15711, stat: Journal Article,

Structure-guided design and immunological characterization of immunogen constructs presenting the HIV-1 gp120 V3 loop on a CTB scaffold
Totrov, M; Jiang, X; Kong, X; Cohen, S; Krachmarov, C; Williams, C; Cardozo, T; Gorny, M; Wang, S; Lu, S; Pinter, A; Zolla-Pazner, S
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105707, year: 2009, vol: 6, page: 115, stat: Journal Article,

Specificity of staphylococcal phage and SaPI DNA packaging as revealed by integrase and terminase mutations
Ubeda, Carles; Olivarez, Nicholas P; Barry, Peter; Wang, Huaibin; Kong, Xiangpeng; Matthews, Avery; Tallent, Sandra M; Christie, Gail E; Novick, Richard P
2009 Apr;72(1):98-108, Molecular microbiology
SaPI1 and SaPIbov1 are chromosomal pathogenicity islands in Staphylococcus aureus that carry tst and other superantigen genes. They are induced to excise and replicate by certain phages, are efficiently encapsidated in SaPI-specific small particles composed of phage virion proteins and are transferred at very high frequencies. In this study, we have analysed three SaPI genes that are important for the phage-SaPI interaction, int (integrase) terS (phage terminase small subunit homologue) and pif (phage interference function). SaPI1 int is required for SaPI excision, replication and packaging in a donor strain, and is required for integration in a recipient. A SaPI1 int mutant, following phage induction, produces small SaPI-specific capsids which are filled with partial phage genomes. SaPIbov1 DNA is efficiently packaged into full-sized phage heads as well as into SaPI-specific small ones, whereas SaPI1 DNA is found almost exclusively in the small capsids. TerS, however, determines DNA packaging specificity but not the choice of large versus small capsids. This choice is influenced by SaPIbov1 gene 12, which prevents phage DNA packaging into small capsids, and which is also primarily responsible for interference by SaPIbov1 with phage reproduction
— id: 97845, year: 2009, vol: 72, page: 98, stat: Journal Article,

Uropathogenic E. coli adhesin-induced host cell receptor conformational changes: implications in transmembrane signaling transduction
Wang, Huaibin; Min, Guangwei; Glockshuber, Rudi; Sun, Tung-Tien; Kong, Xiang-Peng
2009 Sep 18;392(2):352-361, Journal of molecular biology
Urinary tract infection is the second most common infectious disease and is caused predominantly by type 1-fimbriated uropathogenic Escherichia coli (UPEC). UPEC initiates infection by attaching to uroplakin (UP) Ia, its urothelial surface receptor, via the FimH adhesins capping the distal end of its fimbriae. UP Ia, together with UP Ib, UP II, and UP IIIa, forms a 16-nm receptor complex that is assembled into hexagonally packed, two-dimensional crystals (urothelial plaques) covering >90% of the urothelial apical surface. Recent studies indicate that FimH is the invasin of UPEC as its attachment to the urothelial surface can induce cellular signaling events including calcium elevation and the phosphorylation of the UP IIIa cytoplasmic tail, leading to cytoskeletal rearrangements and bacterial invasion. However, it remains unknown how the binding of FimH to the UP receptor triggers a signal that can be transmitted through the highly impermeable urothelial apical membrane. We show here by cryo-electron microscopy that FimH binding to the extracellular domain of UP Ia induces global conformational changes in the entire UP receptor complex, including a coordinated movement of the tightly bundled transmembrane helices. This movement of the transmembrane helix bundles can cause a corresponding lateral translocation of the UP cytoplasmic tails, which can be sufficient to trigger downstream signaling events. Our results suggest a novel pathogen-induced transmembrane signal transduction mechanism that plays a key role in the initial stages of UPEC invasion and receptor-mediated bacterial invasion in general
— id: 101952, year: 2009, vol: 392, page: 352, stat: Journal Article,

Uroplakins in urothelial biology, function, and disease
Wu, Xue-Ru; Kong, Xiang-Peng; Pellicer, Angel; Kreibich, Gert; Sun, Tung-Tien
2009 Jun;75(11):1153-1165, Kidney international
Urothelium covers the inner surfaces of the renal pelvis, ureter, bladder, and prostatic urethra. Although morphologically similar, the urothelia in these anatomic locations differ in their embryonic origin and lineages of cellular differentiation, as reflected in their different uroplakin content, expandability during micturition, and susceptibility to chemical carcinogens. Previously thought to be an inert tissue forming a passive barrier between the urine and blood, urothelia have recently been shown to have a secretory activity that actively modifies urine composition. Urothelial cells express a number of ion channels, receptors, and ligands, enabling them to receive and send signals and communicate with adjoining cells and their broader environment. The urothelial surface bears specific receptors that not only allow uropathogenic E. coli to attach to and invade the bladder mucosa, but also provide a route by which the bacteria ascend through the ureters to the kidney to cause pyelonephritis. Genetic ablation of one or more uroplakin genes in mice causes severe retrograde vesicoureteral reflux, hydronephrosis, and renal failure, conditions that mirror certain human congenital diseases. Clearly, abnormalities of the lower urinary tract can impact the upper tract, and vice versa, through the urothelial connection. In this review, we highlight recent advances in the field of urothelial biology by focusing on the uroplakins, a group of urothelium-specific and differentiation-dependent integral membrane proteins. We discuss these proteins' biochemistry, structure, assembly, intracellular trafficking, and their emerging roles in urothelial biology, function, and pathological processes. We also call attention to important areas where greater investigative efforts are warranted.Kidney International (2009) 75, 1153-1165; doi:10.1038/ki.2009.73; published online 1 April 2009
— id: 98907, year: 2009, vol: 75, page: 1153, stat: Journal Article,

Induction of cross-clade neutralizing antibodies with a prime/boost vaccine strategy focused on a neutralizing epitope
Zolla-Pazner, S; Kong, X; Cardozo, T; Hioe, C; Cohen, S; Jiang, X; Gorny, MK; Totrov, M; Pinter, A; Krachmarov, C; Seaman, MS; Wang, S; Lu, S
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105699, year: 2009, vol: 6, page: 115, stat: Journal Article,

Structural Characterization of Neutralizing Human Anti-V3 Monoclonal Antibodies 3074 and 268-D
Burke, VJ; Kim, S; Williams, C; Gorny, MK; Zolla-Pazner, S; Kong, X
2008 OCT ;24(1):46-46, AIDS research & human retroviruses
— id: 91411, year: 2008, vol: 24, page: 46, stat: Journal Article,

Structure of a small-molecule inhibitor of a DNA polymerase sliding clamp
Georgescu, RE; Yurieva, O; Kim, SS; Kuriyan, J; Kong, XP; O'Donnell, M
2008 AUG 12 ;105(32):11116-11121, Proceedings of the National Academy of Sciences of the United States of America
DNA polymerases attach to the DNA sliding clamp through a common overlapping binding site. We identify a small-molecule compound that binds the protein-binding site in the Escherichia coli beta-clamp and differentially affects the activity of DNA polymerases II, III, and IV. To understand the molecular basis of this discrimination, the cocrystal structure of the chemical inhibitor is solved in complex with beta and is compared with the structures of Pol II, Pot III, and Pol IV peptides bound to beta. The analysis reveals that the small molecule localizes in a region of the clamp to which the DNA polymerases attach in different ways. The results suggest that the small molecule may be useful in the future to probe polymerase function with beta, and that the beta-clamp may represent an antibiotic target
— id: 86811, year: 2008, vol: 105, page: 11116, stat: Journal Article,

Structure of a sliding clamp on DNA
Georgescu, Roxana E; Kim, Seung-Sup; Yurieva, Olga; Kuriyan, John; Kong, Xiang-Peng; O'Donnell, Mike
2008 Jan 11;132(1):43-54, Cell
The structure of the E. coli beta clamp polymerase processivity factor has been solved in complex with primed DNA. Interestingly, the clamp directly binds the DNA duplex and also forms a crystal contact with the ssDNA template strand, which binds into the protein-binding pocket of the clamp. We demonstrate that these clamp-DNA interactions function in clamp loading, perhaps by inducing the ring to close around DNA. Clamp binding to template ssDNA may also serve to hold the clamp at a primed site after loading or during switching of multiple factors on the clamp. Remarkably, the DNA is highly tilted as it passes through the beta ring. The pronounced 22 degrees angle of DNA through beta may enable DNA to switch between multiple factors bound to a single clamp simply by alternating from one protomer of the ring to the other
— id: 75445, year: 2008, vol: 132, page: 43, stat: Journal Article,

Immunoglobulin Gene Usage by Neutralizing Human Anti-V3 HIV-1 Monoclonal Antibodies Derived from Clade B and Non-B HIV-1 Infected Individuals
Gorny, MK; Wang, X; Jiang, X; Williams, C; Volsky, B; Revesz, K; Witover, B; Krachmarov, C; Pinter, A; Nadas, A; Zolla-Pazner, S; Kong, X
2008 OCT ;24(1):46-46, AIDS research & human retroviruses
— id: 91412, year: 2008, vol: 24, page: 46, stat: Journal Article,

Structural Basis of the Antibody-Antigen Interaction in Human Anti-V3 HIV-1 Monoclonal Antibodies
Jiang, X; Burke, V; Williams, C; Gorny, MK; Zolla-Pazner, S; Kong, X
2008 OCT ;24(1):11-11, AIDS research & human retroviruses
— id: 91408, year: 2008, vol: 24, page: 11, stat: Journal Article,

A Pachygyria-causing {alpha}-Tubulin Mutation Results in Inefficient Cycling with CCT and a Deficient Interaction with TBCB
Tian, Guoling; Kong, Xiang-Peng; Jaglin, Xavier H; Chelly, Jamel; Keays, David; Cowan, Nicholas J
2008 Mar;19(3):1152-1161, Molecular biology of the cell
The agyria (lissencephaly)/pachygyria phenotypes are catastrophic developmental diseases characterized by abnormal folds on the surface of the brain and disorganized cortical layering. In addition to mutations in at least four genes-LIS1, DCX, ARX and RELN-mutations in a human alpha-tubulin gene, TUBA1A, have recently been identified that cause these diseases. Here, we show that one such mutation, R264C, leads to a diminished capacity of de novo tubulin heterodimer formation. We identify the mechanisms that contribute to this defect. First, there is a reduced efficiency whereby quasinative alpha-tubulin folding intermediates are generated via ATP-dependent interaction with the cytosolic chaperonin CCT. Second, there is a failure of CCT-generated folding intermediates to stably interact with TBCB, one of the five tubulin chaperones (TBCA-E) that participate in the pathway leading to the de novo assembly of the tubulin heterodimer. We describe the behavior of the R264C mutation in terms of its effect on the structural integrity of alpha-tubulin and its interaction with TBCB. In spite of its compromised folding efficiency, R264C molecules that do productively assemble into heterodimers are capable of copolymerizing into dynamic microtubules in vivo. The diminished production of TUBA1A tubulin in R264C individuals is consistent with haploinsufficiency as a cause of the disease phenotype
— id: 78375, year: 2008, vol: 19, page: 1152, stat: Journal Article,

Characteristics of the phagocytic cup induced by uropathogenic Escherichia coli
Wang, Huaibin; Liang, Feng-Xia; Kong, Xiang-Peng
2008 Jun;56(6):597-604, Journal of histochemistry & cytochemistry
Uropathogenic Escherichia coli invade the urothelial umbrella cells by using the zipper mechanism. However, the details of the early events of this invasion, such as the formation of the phagocytic cup, are not yet well understood. We show here, using thin section electron microscopy and immunogold labeling, that the plasma membrane curves around the bacterial surface in the phagocytic cup. There exists a uniform gap between the bacterium and the urothelial membrane, and actin filaments are present in the phagocytic cup. We suggest that the action-reaction between the mechanical forces generated by pilus retraction of the bacterium and the actin polymerization in the urothelial cell plays a role in maintaining the phagocytic cup. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials
— id: 79139, year: 2008, vol: 56, page: 597, stat: Journal Article,

Atomic force microscopy of mammalian urothelial surface
Kreplak, L; Wang, H; Aebi, U; Kong, XP
2007 NOV 23 ;374(2):365-373, Journal of molecular biology
The mammalian urothelium apical surface plays important roles in bladder physiology and diseases, and it provides a unique morphology for ultrastructural studies. Atomic force microscopy (AFM) is an emerging tool for studying the architecture and dynamic properties of biomolecular structures under near-physiological conditions. However, AFM imaging of soft tissues remains a challenge because of the lack of efficient methods for sample stabilization. Using a porous nitrocellulose membrane as the support, we were able to immobilize large pieces of soft mouse bladder tissue, thus enabling us to carry out the first AFM investigation of the mouse urothelial surface. The submicrometer-resolution AFM images revealed many details of the surface features, including the geometry of the urothelial plaques that cover the entire surface and the membrane interdigitation at the cell borders. This interdigitation creates a membrane zipper, likely contributing to the barrier function of the urothelium. In addition, we were able to image the intracellular bacterial communities of type 1-fimbriated bacteria grown between the intermediate filament bundles of the umbrella cells, shedding light on the bacterial colonization of the urothelium. (c) 2007 Elsevier Ltd. All rights reserved
— id: 75420, year: 2007, vol: 374, page: 365, stat: Journal Article,

Selective enrichment and fractionation of phosphopeptides from peptide mixtures by isoelectric focusing after methyl esterification
Xu, Chong-Feng; Wang, Huaibin; Li, Daming; Kong, Xiang-Peng; Neubert, Thomas A
2007 Mar 1;79(5):2007-2014, Analytical chemistry
We have developed a new strategy to enrich and fractionate phosphopeptides from peptide mixtures based on the difference in their isoelectric points (pIs) after methyl esterification. After isoelectric focusing (IEF) of a methylated tryptic digest of a mixture of alpha-S-casein and beta-casein, phosphopeptides were selectively enriched at acidic and neutral pHs while nonphosphopeptides left the focusing gel because their pIs are higher than the upper limit of the immobilized pH gradient. We wrote a web-based program, pIMethylation, to predict the pIs for peptides with and without methyl esterification. Theoretical calculations using pIMethylation indicated that methylated phosphopeptides and non-phosphopeptides can be grouped on the basis of the number of phosphate groups and basic residues in each peptide. Our IEF results were consistent with theoretical pIs of methylated peptides calculated by pIMethylation. We also showed that 2,6-dihydroxy-acetophenone is superior to 2,5-dihydroxybenzoic acid as a matrix for MALDI Q-TOF MS of methylated phosphopeptides in both positive and negative ion modes
— id: 71393, year: 2007, vol: 79, page: 2007, stat: Journal Article,

pH-dependent conformational switch activates the inhibitor of transcription elongation
Laptenko, O; Kim, SS; Lee, J; Starodubtseva, M; Cava, F; Berenguer, J; Kong, XP; Borukhov, S
2006 MAY 17 ;25(10):2131-2141, EMBO journal
Gfh1, a transcription factor from Thermus thermophilus, inhibits all catalytic activities of RNA polymerase ( RNAP). We characterized the Gfh1 structure, function and possible mechanism of action and regulation. Gfh1 inhibits RNAP by competing with NTPs for coordinating the active site Mg2+ ion. This coordination requires at least two aspartates at the tip of the Gfh1 N-terminal coiled-coil domain (NTD). The overall structure of Gfh1 is similar to that of the Escherichia coli transcript cleavage factor GreA, except for the flipped orientation of the C-terminal domain (CTD). We show that depending on pH, Gfh1-CTD exists in two alternative orientations. At pH above 7, it assumes an inactive 'flipped' orientation seen in the structure, which prevents Gfh1 from binding to RNAP. At lower pH, Gfh1-CTD switches to an active 'Gre-like' orientation, which enables Gfh1 to bind to and inhibit RNAP
— id: 64273, year: 2006, vol: 25, page: 2131, stat: Journal Article,

Structural basis for tetraspanin functions as revealed by the cryo-EM structure of uroplakin complexes at 6-A resolution
Min, Guangwei; Wang, Huaibin; Sun, Tung-Tien; Kong, Xiang-Peng
2006 Jun 19;173(6):975-983, Journal of cell biology
Tetraspanin uroplakins (UPs) Ia and Ib, together with their single-spanning transmembrane protein partners UP II and IIIa, form a unique crystalline 2D array of 16-nm particles covering almost the entire urothelial surface. A 6 A-resolution cryo-EM structure of the UP particle revealed that the UP tetraspanins have a rod-shaped structure consisting of four closely packed transmembrane helices that extend into the extracellular loops, capped by a disulfide-stabilized head domain. The UP tetraspanins form the primary complexes with their partners through tight interactions of the transmembrane domains as well as the extracellular domains, so that the head domains of their tall partners can bridge each other at the top of the heterotetramer. The secondary interactions between the primary complexes and the tertiary interaction between the 16-nm particles contribute to the formation of the UP tetraspanin network. The rod-shaped tetraspanin structure allows it to serve as stable pilings in the lipid sea, ideal for docking partner proteins to form structural/signaling networks
— id: 67387, year: 2006, vol: 173, page: 975, stat: Journal Article,

An arsenite-inducible 19S regulatory particle-associated protein adapts proteasomes to proteotoxicity
Stanhill, Ariel; Haynes, Cole M; Zhang, Yuhong; Min, Guangwei; Steele, Matthew C; Kalinina, Juliya; Martinez, Enid; Pickart, Cecile M; Kong, Xiang-Peng; Ron, David
2006 Sep 15;23(6):875-885, Molecular cell
Protein misfolding caused by exposure to arsenite is associated with transcriptional activation of the AIRAP gene. We report here that AIRAP is an arsenite-inducible subunit of the proteasome's 19S cap that binds near PSMD2 at the 19S base. Compared to the wild-type, knockout mouse cells or C. elegans lacking AIRAP accumulate more polyubiquitylated proteins and exhibit higher levels of stress when exposed to arsenite, and proteasomes isolated from arsenite-treated AIRAP knockout cells are relatively impaired in substrate degradation in vitro. AIRAP's association with the 19S cap reverses the stabilizing affect of ATP on the 26S proteasome during particle purification, and AIRAP-containing proteasomes, though constituted of 19S and 20S subunits, acquire features of hybrid proteasomes with both 19S and 11S regulatory caps. These features include enhanced cleavage of peptide substrates and suggest that AIRAP adapts the cell's core protein degradation machinery to counteract proteotoxicity induced by an environmental toxin
— id: 69075, year: 2006, vol: 23, page: 875, stat: Journal Article,

Integrity of all four transmembrane domains of the tetraspanin uroplakin Ib is required for its exit from the ER
Tu, Liyu; Kong, Xiang-Peng; Sun, Tung-Tien; Kreibich, Gert
2006 Dec 15;119(Pt 24):5077-5086, Journal of cell science
The surface of the mammalian urinary bladder is covered by a crystalline, asymmetric unit membrane (AUM) structure that contains the four major uroplakins (UPs): Ia, Ib, II and IIIa. UPIa and UPIb belong to the family of tetraspanins. Although UPIa and UPIb are structurally conserved, only UPIb could exit from the endoplasmic reticulum (ER) and reach the cell surface when expressed alone in 293T cells. Modifications of the large extracellular loop of UPIb, such as mutation of the N-glycosylation site or the cysteines involved in the formation of three disulfide bridges, or exchanging the large luminal loop of UPIb with that of UPIa did not affect the ability of UPIb to reach the cell surface. However, modifications of any of the four transmembrane domains of UPIb led to ER retention, suggesting that the proper formation of helical bundles consisting of the tetraspanin transmembrane domains is a prerequisite for UPIb to exit from the ER. Results of sedimentation analysis suggested that aggregate formation is a mechanism for ER retention
— id: 71581, year: 2006, vol: 119, page: 5077, stat: Journal Article,

Distinct glycan structures of uroplakins Ia and Ib: structural basis for the selective binding of FimH adhesin to uroplakin Ia
Xie, Bo; Zhou, Ge; Chan, Shiu-Yung; Shapiro, Ellen; Kong, Xiang-Peng; Wu, Xue-Ru; Sun, Tung-Tien; Costello, Catherine E
2006 May 26;281(21):14644-14653, Journal of biological chemistry
Although it has been shown that mouse uroplakin (UP) Ia, a major glycoprotein of urothelial apical surface, can serve as the receptor for the FimH lectin adhesin of type 1-fimbriated Escherichia coli, the organism that causes a great majority of urinary tract infections, the glycan structure of this native receptor was unknown. Using a sensitive approach that combines in-gel glycosidase and protease digestions, permethylation of released glycans, and mass spectrometry, we have elucidated for the first time the native glycoform structures of the mouse UPIa receptor and those of its non-binding homolog, UPIb, and have determined the glycosylation site occupancy. UPIa presents a high level of terminally exposed mannose residues (located on Man(6)GlcNAc(2) to Man(9)GlcNAc(2)) that are capable of specifically interacting with FimH. We have shown that this property is conserved not only in the mouse uroplakins but also in cattle and, even more importantly, in human UPIa, thus establishing the concept that UPIa is a major urothelial receptor in humans and other mammals for the mannose-specific FimH variant. In contrast, our results indicate that most terminally exposed glycans of mouse UPIb are non-mannose residues, thus explaining the failure of FimH to bind to this UPIb. In cattle, on the other hand, complex carbohydrates constituted only about 20% of the UPIb N-linked glycans. Human UPIa contained exclusively high mannose glycans, and human UPIb contained only complex glycans. The drastically different carbohydrate processing of the UPIa and UPIb proteins, two closely related members of the tetraspanin family, may reflect differences in their folding and masking due to their interactions with their associated proteins, UPII and UPIIIa, respectively. Results from this study shed light on the molecular pathogenesis of urinary tract infections and may aid in the design of glyco-mimetic inhibitors for preventing and treating this disease
— id: 66476, year: 2006, vol: 281, page: 14644, stat: Journal Article,

AMPA receptor assembly determined by Q/R editing
Greger, I. H.; Khatri, L.; Kong, X.; Ziff, E. B.
2003 ;2003:?-?, Society for Neuroscience Abstract Viewer & Itinerary Planner
AMPA receptors are tetrameric cation channels that mediate the majority of fast excitatory transmission in the brain. Four subunits, GluR1-4 (or A-D) assemble in various stoichiometries, resulting in a spectrum of functionally distinct channels. Rules that govern assembly are largely unknown. The majority of AMPARs contain GluR2, which dominates transmission properties via Arg607, introduced into the pore loop by RNA editing (at the Q/R-site). Here we report that Arg607 also determines AMPAR assembly. Sedimentation analysis reveals that edited GluR2-R channels remain incompletely assembled and ER-retained, whereas unedited GluR2-Q channels readily tetramerize and exit from the ER, in neurons and HeLa cells. Mutagenesis reveals that the structure of the pore loop affects tetramerization. Therefore, by modulating a critical assembly surface, Q/R-editing determines AMPAR assembly and subunit stoichiometry
— id: 92627, year: 2003, vol: 2003, page: ?, stat: Journal Article,

AMPA receptor tetramerization is mediated by Q/R editing
Greger, Ingo H; Khatri, Latika; Kong, Xiangpeng; Ziff, Edward B
2003 Nov 13;40(4):763-774, Neuron
AMPA-type glutamate receptors (AMPARs) play a major role in excitatory synaptic transmission and plasticity. Channel properties are largely dictated by their composition of the four subunits, GluR1-4 (or A-D). Here we show that AMPAR assembly and subunit stoichiometry are determined by RNA editing in the pore loop. We demonstrate that editing at the GluR2 Q/R site regulates AMPAR assembly at the step of tetramerization. Specifically, edited R subunits are largely unassembled and ER retained, whereas unedited Q subunits readily tetramerize and traffic to synapses. This assembly mechanism restricts the number of the functionally critical R subunits in AMPAR tetramers. Therefore, a single amino acid residue affects channel composition and, in turn, controls ion conduction through the majority of AMPARs in the brain
— id: 44855, year: 2003, vol: 40, page: 763, stat: Journal Article,

Structural basis of urothelial permeability barrier function as revealed by Cryo-EM studies of the 16 nm uroplakin particle
Min, Guangwei; Zhou, Ge; Schapira, Matthieu; Sun, Tung-Tien; Kong, Xiang-Peng
2003 Oct 15;116(Pt 20):4087-4094, Journal of cell science
The apical surface of terminally differentiated mammalian urothelial umbrella cells is covered by numerous plaques consisting of two-dimensional (2D) crystals of hexagonally packed 16 nm uroplakin particles, and functions as a remarkable permeability barrier. To determine the structural basis of this barrier function, we generated, by electron cryo microscopy, a projection map of the isolated mouse urothelial plaques at 7 A and a 3D structure at 10 A resolution. Our results indicate that each 16 nm particle has a central 6 nm lipid-filled 'hole' surrounded by 6 inverted U-shaped subunits, each consisting of an inner and an outer subdomain connected via a distal joint. The transmembrane portion of each subdomain can fit about 5 helices. This finding, coupled with our STEM and EM data, suggests that uroplakin pairs Ia/II and Ib/III are associated with the inner and outer subdomains, respectively. Since the inner subdomains interconnect to form a ring, which can potentially segregate the lipids of the central hole from those outside, the 2D crystalline uroplakin network may impose an organized state and a severely restricted freedom of movement on the lipid components, thus reducing membrane fluidity and contributing to the barrier function of urothelial plaques. Our finding that distinct uroplakin substructures are in contact with the cytoplasmic and exoplasmic leaflets of the plaque suggests that the two leaflets may have different lipid composition and contribute asymmetrically to the barrier function. We propose that the crystalline lattice structure of uroplakin, through its interactions with specialized lipids, plays a major role in the remarkable permeability barrier function of urothelial apical surface. Our results also have implications for the transmembrane signal transduction in urothelial cells as induced by the binding of uropathogenic E. coli to its uroplakin receptor
— id: 39072, year: 2003, vol: 116, page: 4087, stat: Journal Article,

Urothelial plaque as a unique model of tetraspanin network and lipid raft
Min, G; Kim, S; Kong, X
2002 NOV ;13(4):143A-143A, Molecular biology of the cell
— id: 37186, year: 2002, vol: 13, page: 143A, stat: Journal Article,

Localization of uroplakin Ia, the urothelial receptor for bacterial adhesin FimH, on the six inner domains of the 16 nm urothelial plaque particle
Min, Guangwei; Stolz, Martin; Zhou, Ge; Liang, Fengxia; Sebbel, Peter; Stoffler, Daniel; Glockshuber, Rudi; Sun, Tung-Tien; Aebi, Ueli; Kong, Xiang-Peng
2002 Apr 12;317(5):697-706, Journal of molecular biology
The binding of uropathogenic Escherichia coli to the urothelial surface is a critical initial event for establishing urinary tract infection, because it prevents the bacteria from being removed by micturition and it triggers bacterial invasion as well as host cell defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium and its urothelial receptor, uroplakin Ia (UPIa). To localize the UPIa receptor on the 16 nm particles that form two-dimensional crystals of asymmetric unit membrane (AUM) covering >90 % of the apical urothelial surface, we constructed a 15 A resolution 3-D model of the mouse 16 nm AUM particle by negative staining and electron crystallography. Similar to previous lower-resolution models of bovine and pig AUM particles, the mouse 16 nm AUM particle consists of six inner and six outer domains that are interconnected to form a twisted ribbon-like structure. Treatment of urothelial plaques with 0.02-0.1 % (v/v) Triton X-100 allowed the stain to penetrate into the membrane, revealing parts of the uroplakin transmembrane moiety with an overall diameter of 14 nm, which was much bigger than the 11 nm value determined earlier by quick-freeze deep-etch. Atomic force microscopy of native, unfixed mouse and bovine urothelial plaques confirmed the overall structure of the luminal 16 nm AUM particle that was raised by 6.5 nm above the luminal membrane surface and, in addition, revealed a circular, 0.5 nm high, cytoplasmic protrusion of approximately 14 nm diameter. Finally, a difference map calculated from the mouse urothelial plaque images collected in the presence and absence of recombinant bacterial FimH/FimC complex revealed the selective binding of FimH to the six inner domains of the 16 nm AUM particle. These results indicate that the 16 nm AUM particle is anchored by a approximately 14 nm diameter transmembrane stalk, and suggest that bacterial binding to UPIa that resides within the six inner domains of the 16 nm AUM particle may preferentially trigger transmembrane signaling involved in bacterial invasion and host cell defense
— id: 59002, year: 2002, vol: 317, page: 697, stat: Journal Article,

Organization of uroplakin subunits: transmembrane topology, pair formation and plaque composition
Liang FX; Riedel I; Deng FM; Zhou G; Xu C; Wu XR; Kong XP; Moll R; Sun TT
2001 Apr 1;355(Pt 1):13-18, Biochemical journal
The apical surfaces of urothelial cells are almost entirely covered with plaques consisting of crystalline, hexagonal arrays of 16 nm uroplakin particles. Although all four uroplakins, when SDS-denatured, can be digested by chymotrypsin, most uroplakin domains in native urothelial plaques are resistant to the enzyme, suggesting a tightly packed structure. The only exception is the C-terminal, cytoplasmic tail of UPIII (UPIII) which is highly susceptible to proteolysis, suggesting a loose configuration. When uroplakins are solubilized with 2% octylglucoside and fractionated with ion exchangers, UPIa and UPII were bound as a complex by a cation exchanger, whereas UPIb and UPIII were bound by an anion exchanger. This result is consistent with the fact that UPIa and UPIb are cross-linked to UPII and UPIII, respectively, and suggests that the four uroplakins form two pairs consisting of UPIa/II and UPIb/III. Immunogold labelling using a new mouse monoclonal antibody, AU1, revealed that UPIII is present in all urothelial plaques, indicating that the two uroplakin pairs are not segregated into two different types of urothelial plaque and that all plaques must have a similar uroplakin composition. Taken together, these results indicate that uroplakins form a tightly packed structure, that the four uroplakins interact specifically forming two pairs, and that both uroplakin pairs are required for normal urothelial plaque formation
— id: 21231, year: 2001, vol: 355, page: 13, stat: Journal Article,

Uroplakin Ia is the urothelial receptor for uropathogenic Escherichia coli: evidence from in vitro FimH binding
Zhou G; Mo WJ; Sebbel P; Min G; Neubert TA; Glockshuber R; Wu XR; Sun TT; Kong XP
2001 Nov;114(Pt 22):4095-4103, Journal of cell science
The binding of uropathogenic Escherichia coli to the urothelial surface is a crucial initial event for establishing urinary tract infection because it allows the bacteria to gain a foothold on the urothelial surface, thus preventing them from being removed by micturition. In addition, it triggers bacterial invasion as well as host urothelial defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium, a filamentous attachment apparatus, and its urothelial receptor. We have prepared a biotinylated, recombinant FimH-FimC adhesin:chaperone complex and used it to identify its mouse urothelial receptor. The FimH-FimC complex binds specifically to a single 24 kDa major mouse urothelial plaque protein, which we identified as uroplakin Ia by mass spectrometry, cDNA cloning and immunoreactivity. The terminal mannosyl moieties on Asn-169 of uroplakin Ia are responsible for FimH as well as concanavalin A binding. Although FimH binds to uroplakin Ia with only moderate strength (K(d) approximately 100 nM between pH 4 and 9), the binding between multiple fimbriae of a bacterium and the crystalline array of polymerized uroplakin receptors should achieve high avidity and stable bacterial attachment. The FimH-FimC complex binds preferentially to the mouse urothelial umbrella cells in a pattern similar to uroplakin staining. Our results indicate that the structurally related uroplakins Ia and Ib are glycosylated differently, that uroplakin Ia serves as the urothelial receptor for the type 1-fimbriated E. coli, and that the binding of uropathogenic bacteria to uroplakin Ia may play a key role in mediating the urothelial responses to bacterial attachment
— id: 26903, year: 2001, vol: 114, page: 4095, stat: Journal Article,

Crystal structure of human stem cell factor: implication for stem cell factor receptor dimerization and activation
Zhang Z; Zhang R; Joachimiak A; Schlessinger J; Kong XP
2000 Jul 5;97(14):7732-7737, Proceedings of the National Academy of Sciences of the United States of America
Stem cell factor (SCF) plays important roles in hematopoiesis and the survival, proliferation, and differentiation of mast cells, melanocytes, and germ cells. SCF mediates its biological effects by binding to and activating a receptor tyrosine kinase designated c-kit or SCF receptor. In this report we describe the 2.3-A crystal structure of the functional core of recombinant human SCF. SCF is a noncovalent homodimer composed of two slightly wedged protomers. Each SCF protomer exhibits an antiparallel four-helix bundle fold. Dimerization is mediated by extensive polar and nonpolar interactions between the two protomers with a large buried surface area. Finally, we have identified a hydrophobic crevice and a charged region at the tail of each protomer that functions as a potential receptor-binding site. On the basis of these observations, a model for SCF small middle dotc-kit complex formation and dimerization is proposed
— id: 11614, year: 2000, vol: 97, page: 7732, stat: Journal Article,

Identification of conserved amino acids in murine B7-1IgV domain critical for CTLA4/CD28:B7 interaction by site-directed mutagenesis: a novel structural model of the binding site
Guo Y; Wu Y; Kong X; Liu Y
1998 Mar;35(4):215-225, Molecular immunology
The B7: CD28/CTLA4 interaction plays a major role in T cell responses. Immune intervention targeted at this interaction has demonstrated a vast potential in enhancing tumor immunity and blocking autoimmunity and transplant rejection. However, the structural basis for this interaction is unclear. While we and others have performed site-directed mutagenesis to define amino acids involved in binding CD28 and CTLA4, these residues are localized in different regions, and it is unlikely for all of them to be directly involved. In addition, the effect of the mutations on the overall conformation of B7 has not been systematically evaluated. In this study, we have carried out site-directed mutagenesis to define the amino acids within B7-1 IgV-like domain which participate B7:CD28/CTLA4 interaction. Four anti-B7-1 mAbs that recognize three independent antigenic epitopes on B7-1 were used to monitor the effect of mutations on the overall conformation of B7-1. Of the five mutations in the IgV domain that we have produced, D113 > A appears to interfere with cell surface expression and/or overall conformation of B7-1. while four others do not significantly affect the overall conformation and cell surface expression of B7-1. Among them, G115 > A and Y91 > A eliminated B7-1 binding to both CD28Ig and CTLA4Ig; our previously reported mutants L109 > A and W88 > A selectively affect the B7-1 binding to either CD28Ig or CTLA4Ig. Structural modeling of B7-1 based on the structure of immunoglobulin revealed that these four and other previously identified critical amino acids in both IgV- and IgC-like domains can form a localized structure
— id: 8032, year: 1998, vol: 35, page: 215, stat: Journal Article,

An adenosine deaminase (ADA) allele contains two newly identified deleterious mutations (Y97C and L106V) that interact to abolish enzyme activity
Jiang C; Hong R; Horowitz SD; Kong X; Hirschhorn R
1997 Dec;6(13):2271-2278, Human molecular genetics
Genetic deficiency of the purine salvage enzyme adenosine deaminase (ADA) results in varying degrees of immunodeficiency, ranging from neonatal onset Severe Combined Immunodeficiency (SCID) to an adult onset immunodeficiency disorder. Multiple different mutations have now been identified in these immunodeficient patients. Additional mutations, initially identified in healthy individuals, abolish ADA in erythrocytes but retain 10-80% of activity in non-erythroid cells ('partial deficiency mutations'). In general, severity of disease correlates inversely with the amount of residual ADA expressed by the mutant enzymes and directly with the accumulation of the toxic metabolites deoxyATP and deoxyadenosine. We report two newly identified mutations (Y97C and L106V), both carried on the same allele of an immunodeficient patient who was diagnosed prenatally and successfully transplanted with haploidentical bone marrow. Based on the ability of mutant cDNAs to express ADA in vitro , the L106V mutation resulted in activity similar to 'partial' mutations (30% of normal) while the Y97C mutation resulted in detectable but markedly reduced activity (1.5% of normal). However, the presence of both mutations on the same allele virtually abolished detectable enzyme activity. Analysis of the crystallographic structure of ADA to understand the marked deleterious effect of the Y97C mutation suggested a previously unappreciated role of salt bridges in the catalytic mechanism of ADA. The patient was also heteroallelic for a previously described deletion of the promoter and exon 1. Testing of additional patients in whom we had not identified a mutation on the second allele revealed presence of this deletion in three of four patients tested. This deletion is therefore relatively common, accounting for 10% of almost 100 chromosomes studied by this and other laboratories, but is easily missed by currently used methods of mutation detection. Lastly, the finding of two mutations on the same allele that interact to reduce residual enzyme function emphasizes hazards in evaluating potential genotype-phenotype correlations in individuals analyzed only for the presence of single specific mutations
— id: 7960, year: 1997, vol: 6, page: 2271, stat: Journal Article,

Crystallization of proliferating cell nuclear antigen (PCNA) from Saccharomyces cerevisiae
Krishna, T S; Fenyo, D; Kong, X P; Gary, S; Chait, B T; Burgers, P; Kuriyan, J
1994 Aug 12;241(2):265-268, Journal of molecular biology
Proliferating cell nuclear antigen (PCNA) is the component of the chromosomal DNA replication machinery in eukaryotic cells that confers high processivity upon DNA polymerase delta and epsilon. It has been proposed that PCNA functions by forming a trimeric complex with a ring-like structure through which DNA is threaded. PCNA from the yeast Saccharomyces cerevisiae has been crystallized in a cubic space group (P2(1)3, a = 121.1 A). Unexpectedly, a mercury derivative of PCNA yields crystals that diffract significantly better than crystals of the unmodified protein (2.4 A and 3.0 A resolution, respectively). Mass spectrometry reveals that the derivative results from the addition of two mercury atoms to the protein. Although crystals of the mercurated protein show evidence of non-isomorphism, the anomalous diffraction signal is strong and phases may be determined by multi-wavelength anomalous diffraction (MAD phasing)
— id: 114136, year: 1994, vol: 241, page: 265, stat: Journal Article,