Unique mechanisms to increase structural stability and enhance antigen binding in nanobodies

IF 4.4 2区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Structure Pub Date : 2025-02-11 DOI:10.1016/j.str.2025.01.019

Natalia E. Ketaren, Peter C. Fridy, Vladimir Malashkevich, Tanmoy Sanyal, Marc Brillantes, Mary K. Thompson, Deena A. Oren, Jeffrey B. Bonanno, Andrej Šali, Steven C. Almo, Brian T. Chait, Michael P. Rout

{"title":"Unique mechanisms to increase structural stability and enhance antigen binding in nanobodies","authors":"Natalia E. Ketaren, Peter C. Fridy, Vladimir Malashkevich, Tanmoy Sanyal, Marc Brillantes, Mary K. Thompson, Deena A. Oren, Jeffrey B. Bonanno, Andrej Šali, Steven C. Almo, Brian T. Chait, Michael P. Rout","doi":"10.1016/j.str.2025.01.019","DOIUrl":null,"url":null,"abstract":"Nanobodies are single domain antibody variants proving themselves to be compelling tools for research, disease diagnostics, and as therapeutics targeting a myriad of disease agents. However, despite this potential, their mechanisms of paratope presentation and structural stabilization have not been fully explored. Here, we show that unlike monoclonal antibodies, a nanobody repertoire maximizes sampling of an antigen surface by binding a single antigen in at least three different orientations, which are correlated with their paratope composition. Structure-guided reengineering of several nanobodies reveals that a single point mutation within the paratope or a highly conserved region of a nanobody’s framework 3 (FR3) can markedly improve antigen affinity, nanobody stability, or both. Conversely, we show the negative impact on antigen affinity when “over-stabilizing” nanobodies. Collectively our results provide a universal strategy to tune a nanobody’s affinity by modifying specific residues that can readily be applied to guide nanobody optimization and functionalization.","PeriodicalId":22168,"journal":{"name":"Structure","volume":"128 1","pages":""},"PeriodicalIF":4.4000,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Structure","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/j.str.2025.01.019","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}

引用次数: 0

Abstract

Nanobodies are single domain antibody variants proving themselves to be compelling tools for research, disease diagnostics, and as therapeutics targeting a myriad of disease agents. However, despite this potential, their mechanisms of paratope presentation and structural stabilization have not been fully explored. Here, we show that unlike monoclonal antibodies, a nanobody repertoire maximizes sampling of an antigen surface by binding a single antigen in at least three different orientations, which are correlated with their paratope composition. Structure-guided reengineering of several nanobodies reveals that a single point mutation within the paratope or a highly conserved region of a nanobody’s framework 3 (FR3) can markedly improve antigen affinity, nanobody stability, or both. Conversely, we show the negative impact on antigen affinity when “over-stabilizing” nanobodies. Collectively our results provide a universal strategy to tune a nanobody’s affinity by modifying specific residues that can readily be applied to guide nanobody optimization and functionalization.

Abstract Image

查看原文

微信好友朋友圈 QQ好友复制链接

本刊更多论文

求助全文

约1分钟内获得全文去求助

来源期刊

Structure 生物-生化与分子生物学

CiteScore

8.90

自引率

1.80%

发文量

155

审稿时长

3-8 weeks

期刊介绍： Structure aims to publish papers of exceptional interest in the field of structural biology. The journal strives to be essential reading for structural biologists, as well as biologists and biochemists that are interested in macromolecular structure and function. Structure strongly encourages the submission of manuscripts that present structural and molecular insights into biological function and mechanism. Other reports that address fundamental questions in structural biology, such as structure-based examinations of protein evolution, folding, and/or design, will also be considered. We will consider the application of any method, experimental or computational, at high or low resolution, to conduct structural investigations, as long as the method is appropriate for the biological, functional, and mechanistic question(s) being addressed. Likewise, reports describing single-molecule analysis of biological mechanisms are welcome. In general, the editors encourage submission of experimental structural studies that are enriched by an analysis of structure-activity relationships and will not consider studies that solely report structural information unless the structure or analysis is of exceptional and broad interest. Studies reporting only homology models, de novo models, or molecular dynamics simulations are also discouraged unless the models are informed by or validated by novel experimental data; rationalization of a large body of existing experimental evidence and making testable predictions based on a model or simulation is often not considered sufficient.