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| - | [[Image:1ohq.jpg|left|200px]]<br /><applet load="1ohq" size="350" color="white" frame="true" align="right" spinBox="true" | |
| - | caption="1ohq, resolution 2.0Å" /> | |
| - | '''CRYSTAL STRUCTURE OF HEL4, A SOLUBLE HUMAN VH ANTIBODY DOMAIN RESISTANT TO AGGREGATION'''<br /> | |
| | | | |
| - | ==Overview== | + | ==Crystal structure of HEL4, a soluble human VH antibody domain resistant to aggregation== |
| - | The antigen binding site of antibodies usually comprises associated heavy, (V(H)) and light (V(L)) chain variable domains, but in camels and llamas, the binding site frequently comprises the heavy chain variable domain only, (referred to as V(HH)). In contrast to reported human V(H) domains, V(HH), domains are well expressed from bacteria and yeast, are readily purified, in soluble form and refold reversibly after heat-denaturation. These, desirable properties have been attributed to highly conserved, substitutions of the hydrophobic residues of V(H) domains, which normally, interact with complementary V(L) domains. Here, we describe the discovery, and characterisation of an isolated human V(H) domain (HEL4) with, properties similar to those of V(HH) domains. HEL4 is highly soluble at, concentrations of > or =3 mM, essentially monomeric and resistant to, aggregation upon thermodenaturation at concentrations as high as 56, microM. However, in contrast to V(HH) domains, the hydrophobic framework, residues of the V(H):V(L) interface are maintained and the only sequence, changes from the corresponding human germ-line segment (V3-23/DP-47) are, located in the loops comprising the complementarity determining regions, (CDRs). The crystallographic structure of HEL4 reveals an unusual feature;, the side-chain of a framework residue (Trp47) is flipped into a cavity, formed by Gly35 of CDR1, thereby increasing the hydrophilicity of the, V(H):V(L) interface. To evaluate the specific contribution of Gly35 to, domain properties, Gly35 was introduced into a V(H) domain with poor, solution properties. This greatly enhanced the recovery of the mutant from, a gel filtration matrix, but had little effect on its ability to refold, reversibly after heat denaturation. Our results confirm the importance of, a hydrophilic V(H):V(L) interface for purification of isolated V(H), domains, and constitute a step towards the design of isolated human V(H), domains with practical properties for immunotherapy. | + | <StructureSection load='1ohq' size='340' side='right'caption='[[1ohq]], [[Resolution|resolution]] 2.00Å' scene=''> |
| | + | == Structural highlights == |
| | + | <table><tr><td colspan='2'>[[1ohq]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1OHQ OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1OHQ FirstGlance]. <br> |
| | + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2Å</td></tr> |
| | + | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1ohq FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1ohq OCA], [https://pdbe.org/1ohq PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1ohq RCSB], [https://www.ebi.ac.uk/pdbsum/1ohq PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1ohq ProSAT]</span></td></tr> |
| | + | </table> |
| | + | == Function == |
| | + | [https://www.uniprot.org/uniprot/HV323_HUMAN HV323_HUMAN] V region of the variable domain of immunoglobulin heavy chains that participates in the antigen recognition (PubMed:24600447). Immunoglobulins, also known as antibodies, are membrane-bound or secreted glycoproteins produced by B lymphocytes. In the recognition phase of humoral immunity, the membrane-bound immunoglobulins serve as receptors which, upon binding of a specific antigen, trigger the clonal expansion and differentiation of B lymphocytes into immunoglobulins-secreting plasma cells. Secreted immunoglobulins mediate the effector phase of humoral immunity, which results in the elimination of bound antigens (PubMed:20176268, PubMed:22158414). The antigen binding site is formed by the variable domain of one heavy chain, together with that of its associated light chain. Thus, each immunoglobulin has two antigen binding sites with remarkable affinity for a particular antigen. The variable domains are assembled by a process called V-(D)-J rearrangement and can then be subjected to somatic hypermutations which, after exposure to antigen and selection, allow affinity maturation for a particular antigen (PubMed:17576170, PubMed:20176268).<ref>PMID:17576170</ref> <ref>PMID:20176268</ref> <ref>PMID:22158414</ref> <ref>PMID:24600447</ref> |
| | + | == Evolutionary Conservation == |
| | + | [[Image:Consurf_key_small.gif|200px|right]] |
| | + | Check<jmol> |
| | + | <jmolCheckbox> |
| | + | <scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/oh/1ohq_consurf.spt"</scriptWhenChecked> |
| | + | <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview03.spt</scriptWhenUnchecked> |
| | + | <text>to colour the structure by Evolutionary Conservation</text> |
| | + | </jmolCheckbox> |
| | + | </jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=1ohq ConSurf]. |
| | + | <div style="clear:both"></div> |
| | + | <div style="background-color:#fffaf0;"> |
| | + | == Publication Abstract from PubMed == |
| | + | The antigen binding site of antibodies usually comprises associated heavy (V(H)) and light (V(L)) chain variable domains, but in camels and llamas, the binding site frequently comprises the heavy chain variable domain only (referred to as V(HH)). In contrast to reported human V(H) domains, V(HH) domains are well expressed from bacteria and yeast, are readily purified in soluble form and refold reversibly after heat-denaturation. These desirable properties have been attributed to highly conserved substitutions of the hydrophobic residues of V(H) domains, which normally interact with complementary V(L) domains. Here, we describe the discovery and characterisation of an isolated human V(H) domain (HEL4) with properties similar to those of V(HH) domains. HEL4 is highly soluble at concentrations of > or =3 mM, essentially monomeric and resistant to aggregation upon thermodenaturation at concentrations as high as 56 microM. However, in contrast to V(HH) domains, the hydrophobic framework residues of the V(H):V(L) interface are maintained and the only sequence changes from the corresponding human germ-line segment (V3-23/DP-47) are located in the loops comprising the complementarity determining regions (CDRs). The crystallographic structure of HEL4 reveals an unusual feature; the side-chain of a framework residue (Trp47) is flipped into a cavity formed by Gly35 of CDR1, thereby increasing the hydrophilicity of the V(H):V(L) interface. To evaluate the specific contribution of Gly35 to domain properties, Gly35 was introduced into a V(H) domain with poor solution properties. This greatly enhanced the recovery of the mutant from a gel filtration matrix, but had little effect on its ability to refold reversibly after heat denaturation. Our results confirm the importance of a hydrophilic V(H):V(L) interface for purification of isolated V(H) domains, and constitute a step towards the design of isolated human V(H) domains with practical properties for immunotherapy. |
| | | | |
| - | ==About this Structure==
| + | Crystal structure of HEL4, a soluble, refoldable human V(H) single domain with a germ-line scaffold.,Jespers L, Schon O, James LC, Veprintsev D, Winter G J Mol Biol. 2004 Apr 2;337(4):893-903. PMID:15033359<ref>PMID:15033359</ref> |
| - | 1OHQ is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1OHQ OCA].
| + | |
| | | | |
| - | ==Reference==
| + | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> |
| - | Crystal structure of HEL4, a soluble, refoldable human V(H) single domain with a germ-line scaffold., Jespers L, Schon O, James LC, Veprintsev D, Winter G, J Mol Biol. 2004 Apr 2;337(4):893-903. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=15033359 15033359]
| + | </div> |
| | + | <div class="pdbe-citations 1ohq" style="background-color:#fffaf0;"></div> |
| | + | == References == |
| | + | <references/> |
| | + | __TOC__ |
| | + | </StructureSection> |
| | [[Category: Homo sapiens]] | | [[Category: Homo sapiens]] |
| - | [[Category: Single protein]] | + | [[Category: Large Structures]] |
| - | [[Category: James, L.C.]] | + | [[Category: James LC]] |
| - | [[Category: Jespers, L.]] | + | [[Category: Jespers L]] |
| - | [[Category: Schon, O.]] | + | [[Category: Schon O]] |
| - | [[Category: Veprintsev, D.]] | + | [[Category: Veprintsev D]] |
| - | [[Category: Winter, G.]] | + | [[Category: Winter G]] |
| - | [[Category: antibody]]
| + | |
| - | [[Category: fv fragment]]
| + | |
| - | [[Category: hel4]]
| + | |
| - | [[Category: immune system]]
| + | |
| - | [[Category: immunoglobulin]]
| + | |
| - | | + | |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri Feb 15 16:34:25 2008''
| + | |
| Structural highlights
Function
HV323_HUMAN V region of the variable domain of immunoglobulin heavy chains that participates in the antigen recognition (PubMed:24600447). Immunoglobulins, also known as antibodies, are membrane-bound or secreted glycoproteins produced by B lymphocytes. In the recognition phase of humoral immunity, the membrane-bound immunoglobulins serve as receptors which, upon binding of a specific antigen, trigger the clonal expansion and differentiation of B lymphocytes into immunoglobulins-secreting plasma cells. Secreted immunoglobulins mediate the effector phase of humoral immunity, which results in the elimination of bound antigens (PubMed:20176268, PubMed:22158414). The antigen binding site is formed by the variable domain of one heavy chain, together with that of its associated light chain. Thus, each immunoglobulin has two antigen binding sites with remarkable affinity for a particular antigen. The variable domains are assembled by a process called V-(D)-J rearrangement and can then be subjected to somatic hypermutations which, after exposure to antigen and selection, allow affinity maturation for a particular antigen (PubMed:17576170, PubMed:20176268).[1] [2] [3] [4]
Evolutionary Conservation
Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf.
Publication Abstract from PubMed
The antigen binding site of antibodies usually comprises associated heavy (V(H)) and light (V(L)) chain variable domains, but in camels and llamas, the binding site frequently comprises the heavy chain variable domain only (referred to as V(HH)). In contrast to reported human V(H) domains, V(HH) domains are well expressed from bacteria and yeast, are readily purified in soluble form and refold reversibly after heat-denaturation. These desirable properties have been attributed to highly conserved substitutions of the hydrophobic residues of V(H) domains, which normally interact with complementary V(L) domains. Here, we describe the discovery and characterisation of an isolated human V(H) domain (HEL4) with properties similar to those of V(HH) domains. HEL4 is highly soluble at concentrations of > or =3 mM, essentially monomeric and resistant to aggregation upon thermodenaturation at concentrations as high as 56 microM. However, in contrast to V(HH) domains, the hydrophobic framework residues of the V(H):V(L) interface are maintained and the only sequence changes from the corresponding human germ-line segment (V3-23/DP-47) are located in the loops comprising the complementarity determining regions (CDRs). The crystallographic structure of HEL4 reveals an unusual feature; the side-chain of a framework residue (Trp47) is flipped into a cavity formed by Gly35 of CDR1, thereby increasing the hydrophilicity of the V(H):V(L) interface. To evaluate the specific contribution of Gly35 to domain properties, Gly35 was introduced into a V(H) domain with poor solution properties. This greatly enhanced the recovery of the mutant from a gel filtration matrix, but had little effect on its ability to refold reversibly after heat denaturation. Our results confirm the importance of a hydrophilic V(H):V(L) interface for purification of isolated V(H) domains, and constitute a step towards the design of isolated human V(H) domains with practical properties for immunotherapy.
Crystal structure of HEL4, a soluble, refoldable human V(H) single domain with a germ-line scaffold.,Jespers L, Schon O, James LC, Veprintsev D, Winter G J Mol Biol. 2004 Apr 2;337(4):893-903. PMID:15033359[5]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Teng G, Papavasiliou FN. Immunoglobulin somatic hypermutation. Annu Rev Genet. 2007;41:107-20. PMID:17576170 doi:http://dx.doi.org/10.1146/annurev.genet.41.110306.130340
- ↑ Schroeder HW Jr, Cavacini L. Structure and function of immunoglobulins. J Allergy Clin Immunol. 2010 Feb;125(2 Suppl 2):S41-52. doi:, 10.1016/j.jaci.2009.09.046. PMID:20176268 doi:http://dx.doi.org/10.1016/j.jaci.2009.09.046
- ↑ McHeyzer-Williams M, Okitsu S, Wang N, McHeyzer-Williams L. Molecular programming of B cell memory. Nat Rev Immunol. 2011 Dec 9;12(1):24-34. doi: 10.1038/nri3128. PMID:22158414 doi:http://dx.doi.org/10.1038/nri3128
- ↑ Lefranc MP. Immunoglobulin and T Cell Receptor Genes: IMGT((R)) and the Birth and Rise of Immunoinformatics. Front Immunol. 2014 Feb 5;5:22. doi: 10.3389/fimmu.2014.00022. eCollection 2014. PMID:24600447 doi:http://dx.doi.org/10.3389/fimmu.2014.00022
- ↑ Jespers L, Schon O, James LC, Veprintsev D, Winter G. Crystal structure of HEL4, a soluble, refoldable human V(H) single domain with a germ-line scaffold. J Mol Biol. 2004 Apr 2;337(4):893-903. PMID:15033359 doi:10.1016/j.jmb.2004.02.013
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