9fww

From Proteopedia

(Difference between revisions)

Current revision

Human NKp30 in complex with a VHH variant

Structural highlights

9fww is a 2 chain structure with sequence from Homo sapiens. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 1.844Å
Ligands:	,
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

NCTR3_HUMAN Cytotoxicity-activating receptor that contributes to the increased efficiency of activated natural killer (NK) cells to mediate tumor cell lysis. Engagement of NCR3 by BAG6 also promotes dendritic cell (DC) maturation, both through killing those DCs that did not properly acquire a mature phenotype, and inducing NK cells to release TNFA and IFNG, which promotes DC maturation.^[1] ^[2] ^[3]

Publication Abstract from PubMed

The humanization of camelid-derived variable domain heavy chain antibodies (VHHs) poses challenges including immunogenicity, stability, and potential reduction of affinity. Critical to this process are complementarity-determining regions (CDRs), Vernier and Hallmark residues, shaping the three-dimensional fold and influencing VHH structure and function. Additionally, the presence of non-canonical disulfide bonds further contributes to conformational stability and antigen binding. In this study, we systematically humanized two camelid-derived VHHs targeting the natural cytotoxicity receptor NKp30. Key structural positions in Vernier and Hallmark regions were exchanged with residues from the most similar human germline sequences. The resulting variants were characterized for binding affinities, yield, and purity. Structural binding modes were elucidated through crystal structure determination and AlphaFold2 predictions, providing insights into differences in binding affinity. Comparative structural and molecular dynamics characterizations of selected variants were performed to rationalize their functional properties and elucidate the role of specific sequence motifs in antigen binding. Furthermore, systematic analyses of next-generation sequencing (NGS) and Protein Data Bank (PDB) data was conducted, shedding light on the functional significance of Hallmark motifs and non-canonical disulfide bonds in VHHs in general. Overall, this study provides valuable insights into the structural determinants governing the functional properties of VHHs, offering a roadmap for their rational design, humanization, and optimization for therapeutic applications.

On the humanization of VHHs: Prospective case studies, experimental and computational characterization of structural determinants for functionality.,Fernandez-Quintero ML, Guarnera E, Musil D, Pekar L, Sellmann C, Freire F, Sousa RL, Santos SP, Freitas MC, Bandeiras TM, Silva MMS, Loeffler JR, Ward AB, Harwardt J, Zielonka S, Evers A Protein Sci. 2024 Nov;33(11):e5176. doi: 10.1002/pro.5176. PMID:39422475^[4]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Pende D, Parolini S, Pessino A, Sivori S, Augugliaro R, Morelli L, Marcenaro E, Accame L, Malaspina A, Biassoni R, Bottino C, Moretta L, Moretta A. Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells. J Exp Med. 1999 Nov 15;190(10):1505-16. PMID:10562324
↑ Vitale M, Della Chiesa M, Carlomagno S, Pende D, Arico M, Moretta L, Moretta A. NK-dependent DC maturation is mediated by TNFalpha and IFNgamma released upon engagement of the NKp30 triggering receptor. Blood. 2005 Jul 15;106(2):566-71. Epub 2005 Mar 22. PMID:15784725 doi:10.1182/blood-2004-10-4035
↑ Simhadri VR, Reiners KS, Hansen HP, Topolar D, Simhadri VL, Nohroudi K, Kufer TA, Engert A, Pogge von Strandmann E. Dendritic cells release HLA-B-associated transcript-3 positive exosomes to regulate natural killer function. PLoS One. 2008;3(10):e3377. doi: 10.1371/journal.pone.0003377. Epub 2008 Oct 13. PMID:18852879 doi:10.1371/journal.pone.0003377
↑ Fernández-Quintero ML, Guarnera E, Musil D, Pekar L, Sellmann C, Freire F, Sousa RL, Santos SP, Freitas MC, Bandeiras TM, Silva MMS, Loeffler JR, Ward AB, Harwardt J, Zielonka S, Evers A. On the humanization of VHHs: Prospective case studies, experimental and computational characterization of structural determinants for functionality. Protein Sci. 2024 Nov;33(11):e5176. PMID:39422475 doi:10.1002/pro.5176

1 Structural highlights
2 Function
3 Publication Abstract from PubMed
4 References

Proteopedia Page Contributors and Editors (what is this?)

OCA

Retrieved from "http://52.214.119.220/wiki/index.php/9fww"

Categories: Homo sapiens | Large Structures | Freire F | Musil D

@@ Line 1: / Line 1: @@
-'''Unreleased structure'''
-The entry 9fww is ON HOLD  until Paper Publication
+==Human NKp30 in complex with a VHH variant==
+<StructureSection load='9fww' size='340' side='right'caption='[[9fww]], [[Resolution|resolution]] 1.84&Aring;' scene=''>
+== Structural highlights ==
+<table><tr><td colspan='2'>[[9fww]] 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=9FWW OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=9FWW 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]] 1.844&#8491;</td></tr>
+<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=FUC:ALPHA-L-FUCOSE'>FUC</scene>, <scene name='pdbligand=NAG:N-ACETYL-D-GLUCOSAMINE'>NAG</scene></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=9fww FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=9fww OCA], [https://pdbe.org/9fww PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=9fww RCSB], [https://www.ebi.ac.uk/pdbsum/9fww PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=9fww ProSAT]</span></td></tr>
+</table>
+== Function ==
+[https://www.uniprot.org/uniprot/NCTR3_HUMAN NCTR3_HUMAN] Cytotoxicity-activating receptor that contributes to the increased efficiency of activated natural killer (NK) cells to mediate tumor cell lysis. Engagement of NCR3 by BAG6 also promotes dendritic cell (DC) maturation, both through killing those DCs that did not properly acquire a mature phenotype, and inducing NK cells to release TNFA and IFNG, which promotes DC maturation.<ref>PMID:10562324</ref> <ref>PMID:15784725</ref> <ref>PMID:18852879</ref>
+<div style="background-color:#fffaf0;">
+== Publication Abstract from PubMed ==
+The humanization of camelid-derived variable domain heavy chain antibodies (VHHs) poses challenges including immunogenicity, stability, and potential reduction of affinity. Critical to this process are complementarity-determining regions (CDRs), Vernier and Hallmark residues, shaping the three-dimensional fold and influencing VHH structure and function. Additionally, the presence of non-canonical disulfide bonds further contributes to conformational stability and antigen binding. In this study, we systematically humanized two camelid-derived VHHs targeting the natural cytotoxicity receptor NKp30. Key structural positions in Vernier and Hallmark regions were exchanged with residues from the most similar human germline sequences. The resulting variants were characterized for binding affinities, yield, and purity. Structural binding modes were elucidated through crystal structure determination and AlphaFold2 predictions, providing insights into differences in binding affinity. Comparative structural and molecular dynamics characterizations of selected variants were performed to rationalize their functional properties and elucidate the role of specific sequence motifs in antigen binding. Furthermore, systematic analyses of next-generation sequencing (NGS) and Protein Data Bank (PDB) data was conducted, shedding light on the functional significance of Hallmark motifs and non-canonical disulfide bonds in VHHs in general. Overall, this study provides valuable insights into the structural determinants governing the functional properties of VHHs, offering a roadmap for their rational design, humanization, and optimization for therapeutic applications.
-Authors: Musil, D., Freire, F.
+On the humanization of VHHs: Prospective case studies, experimental and computational characterization of structural determinants for functionality.,Fernandez-Quintero ML, Guarnera E, Musil D, Pekar L, Sellmann C, Freire F, Sousa RL, Santos SP, Freitas MC, Bandeiras TM, Silva MMS, Loeffler JR, Ward AB, Harwardt J, Zielonka S, Evers A Protein Sci. 2024 Nov;33(11):e5176. doi: 10.1002/pro.5176. PMID:39422475<ref>PMID:39422475</ref>
-Description: Human NKp30 in complex with a VHH variant
+From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
-[[Category: Unreleased Structures]]
+</div>
-[[Category: Freire, F]]
+<div class="pdbe-citations 9fww" style="background-color:#fffaf0;"></div>
-[[Category: Musil, D]]
+== References ==
+<references/>
+__TOC__
+</StructureSection>
+[[Category: Homo sapiens]]
+[[Category: Large Structures]]
+[[Category: Freire F]]
+[[Category: Musil D]]

9fww

From Proteopedia

Current revision

Human NKp30 in complex with a VHH variant

Structural highlights

Function

Publication Abstract from PubMed

References

Contents

Proteopedia Page Contributors and Editors (what is this?)

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