Probability Analysis of Variational Crystallization and Its Application to gp120, The Exterior Envelope Glycoprotein of Type 1 Human Immunodeficiency Virus (HIV-1)

Kwong, Peter D.; Wyatt, Richard T.; Desjardins, Elizabeth; Robinson, James E.; Culp, Jeffrey S.; Hellmig, Brian D.; Sweet, Raymond W.; Sodroski, Joseph; Hendrickson, Wayne A.

doi:10.1074/jbc.274.7.4115

Cited by 107 publications

(81 citation statements)

References 57 publications

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“…The various biochemical manipulations (such as deglycosylation for gp120 and papain digestion to produce Fab 17b), protein purification and ternary complex crystallization are described elsewhere 25 . The best crystals were small needles of cross-section only 30-40 μm.…”

Section: Methods Protein Production Crystallization and Data Collectionmentioning

confidence: 99%

“…We made truncations at termini and variable loops in various combinations with gp120 from various strains, extensively deglycosylated these gp120 variants, and produced complexes with various ligands. A theoretical analysis indicated that the probability of crystal formation is greatly increased by such reduction of surface heterogeneity and trials with multiple variants 25 . After screening almost twenty combinations of gp120 variants and ligands, we obtained crystals 25 of a ternary complex composed of a truncated form of gp120, the N-terminal two domains (DID2) of CD4, and a Fab from the human neutralizing monoclonal antibody 17b (ref.…”

Section: Structure Determinationmentioning

confidence: 99%

See 1 more Smart Citation

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

Kwong

Wyatt

Robinson

et al. 1998

Nature

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2,742

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The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gpl20 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 Å resolution of an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding. The structure reveals a cavity-laden CD4-gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion. Our results provide a framework for understanding the complex biology of HIV entry into cells and should guide efforts to intervene.The human immunodeficiency viruses HIV-1 and HIV-2 and the related simian immunodeficiency viruses (SIV) cause the destruction of CD4 + lymphocytes in their respective hosts, resulting in the development of acquired immunodeficiency syndromeCorrespondence and requests for materials should be addressed to W. A.H. (wayne@convex.hhmi.columbia.edu).Coordinates have been deposited in the Brookhaven Protein Data Bank (accession code 1gc1) and maybe obtained from the authors. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript (AIDS) 1,2 . The entry of HIV into host cells is mediated by the viral envelope glycoproteins, which are organized into oligomeric, probably trimeric spikes displayed on the surface of the virion. These envelope complexes are anchored in the viral membrane by the gp41 transmembrane envelope glycoprotein. The surface of the spike is composed primarily of the exterior envelope glycoprotein, gp120, associated by non-covalent interactions with each subunit of the trimeric gp41 glycoprotein complex 3,4 . Comparison of the gp120 sequences of different primate immunodeficiency viruses identified five variable regions (V1-V5) (ref. 5 ). The first four variable regions form surface-exposed loops that contain disulphide bonds at their bases 6 . The conserved gp120 regions form discontinuous structures important for the interaction with the gp41 ectodomain and with the viral receptors on the target cell. Both conserved and variable gp120 regions are extensively glycosylated 6 . The variability and glycosylation of the gp120 surface probably modulate the immunogenicity and antigenicity of the gp120 glycoprotein, which is the main target for neutralizing antibodies elicited during natural infection 7 .Entry of primate immunodeficiency viruses into the host cell involves the binding of the gp120 envelope glycoprotein to the CD4 glycoprotein, which serves as the primary receptor. The gp120 glycoprotein binds to the most amino-terminal of the four immunoglobulin-like domains of CD4. S...

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Section: Methods Protein Production Crystallization and Data Collectionmentioning

confidence: 99%

Section: Structure Determinationmentioning

confidence: 99%

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

Kwong

Wyatt

Robinson

et al. 1998

Nature

Self Cite

2,742

108

3,142

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“…Q425 Fab was prepared by papain digestion of reduced and alklated IgG following methods described in ref. 19.…”

Section: Methodsmentioning

confidence: 99%

Interfacial metal and antibody recognition

Zhou

Hamer

Hendrickson

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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The unique ligation properties of metal ions are widely exploited by proteins, with approximately one-third of all proteins estimated to be metalloproteins. Although antibodies use various mechanisms for recognition, to our knowledge, none has ever been characterized that uses an interfacial metal. We previously described a family of CD4-reactive antibodies, the archetype being Q425. CD4:Q425 engagement does not interfere with CD4:HIV-1 gp120 envelope glycoprotein binding, but it blocks subsequent steps required for viral entry. Here, we use surface-plasmon resonance to show that Q425 requires calcium for recognition of CD4. Specifically, Q425 binding of calcium resulted in a 55,000-fold enhancement in affinity for CD4. X-ray crystallographic analyses of Q425 in the presence of Ca 2؉ , Ba 2؉ , or EDTA revealed an exposed metal-binding site, partially coordinated by five atoms contributed from four antibody complementarity-determining regions. The results suggest that Q425 recognition of CD4 involves direct ligation of antigen by the Q425-held calcium, with calcium binding each ligating atom of CD4 with Ϸ1.5 kcal͞mol of binding energy. This energetic contribution, which is greater than that from a typical protein atom, demonstrates how interfacial metal ligation can play a unique role in antigen recognition.antibody Q425 ͉ CD4 ͉ crystal structure ͉ HIV T he humoral immune system employs various mechanisms to generate highly specific antibody (Ab) recognition. Starting from a combinatorial array with a limited number of components with moderate affinity (10 Ϫ6 M), it rapidly evolves high affinity (10 Ϫ9 M) (reviewed in refs. 1-3). Recent structural and biochemical studies of Ab-antigen interactions have revealed several tricks that can augment this process including domain swapping (4), Tyr sulfation (5, 6), and nonspecific interaction with membrane (7,8).One commonly observed strategy for protein recognition, direct metal ligation, has not been observed with Abs. This absence is perhaps surprising in light of the unique coordination properties of metal ions and the wide exploitation observed throughout nature of the special properties of metals: approximately one-third of structurally characterized proteins contain metals, and it is estimated that about the same proportion of all proteins are metalloproteins (reviewed in refs. 9 and 10).Abs can distinguish conformational alterations induced by metals in antigens and have been designed or selected to bind metals by direct ligation (11) or indirectly through metal-binding cofactors (12-15). Many of the latter cases of ''designed'' metal binders have involved ''catalytic'' Abs (16). Despite the eclectic variety of Abs described to date, to our knowledge, none had been observed in which the Ab contributes directly to the ligand-binding sphere of the metal, with the metal also binding directly to the antigen.In investigating the CD4-reactive Ab Q425, we were surprised to find that its binding was affected by EDTA. Because CD4 was not known to bind metal ions and the s...

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“…Removing disordered regions may thus improve homogeneity. The energetics and kinetics of protein crystallization may be facilitated by selective deletion of these unstructured sequences (5). Even those proteins that readily crystallize can suffer from poor diffraction, and it is likely that disorder plays a significant role.…”

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confidence: 99%

Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS

Pantazatos

Kim

Klock

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

142

109

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Crystallographic efforts often fail to produce suitably diffracting protein crystals. Unstructured regions of proteins play an important role in this problem and considerable advantage can be gained in removing them. We have developed a number of enhancements to amide hydrogen͞high-throughput and high-resolution deuterium exchange MS (DXMS) technology that allow rapid identification of unstructured regions in proteins. To demonstrate the utility of this approach for improving crystallization success, DXMS analysis was attempted on 24 Thermotoga maritima proteins with varying crystallization and diffraction characteristics. Data acquisition and analysis for 21 of these proteins was completed in 2 weeks and resulted in the localization and prediction of several unstructured regions within the proteins. When compared with those targets of known structure, the DXMS method correctly localized even small regions of disorder. DXMS analysis was then correlated with the propensity of such targets to crystallize and was further used to define truncations that improved crystallization. Truncations that were defined solely on DXMS analysis demonstrated greatly improved crystallization and have been used for structure determination. This approach represents a rapid and generalized method that can be applied to structural genomics or other targets in a high-throughput manner.

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Probability Analysis of Variational Crystallization and Its Application to gp120, The Exterior Envelope Glycoprotein of Type 1 Human Immunodeficiency Virus (HIV-1)

Cited by 107 publications

References 57 publications

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

Interfacial metal and antibody recognition

Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS

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