Quantitative mapping of binding specificity landscapes for homologous targets by using a high-throughput method

Author:

Aharon Lidan1,Aharoni Shay-Lee1,Radisky Evette S.2,Papo Niv1ORCID

Affiliation:

1. Department of Biotechnology Engineering and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

2. Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville 32224, Florida, U.S.A

Abstract

To facilitate investigations of protein–protein interactions (PPIs), we developed a novel platform for quantitative mapping of protein binding specificity landscapes, which combines the multi-target screening of a mutagenesis library into high- and low-affinity populations with sophisticated next-generation sequencing analysis. Importantly, this method generates accurate models to predict affinity and specificity values for any mutation within a protein complex, and requires only a few experimental binding affinity measurements using purified proteins for calibration. We demonstrated the utility of the approach by mapping quantitative landscapes for interactions between the N-terminal domain of the tissue inhibitor of metalloproteinase 2 (N-TIMP2) and three matrix metalloproteinases (MMPs) having homologous structures but different affinities (MMP-1, MMP-3, and MMP-14). The binding landscapes for N-TIMP2/MMP-1 and N-TIMP2/MMP-3 showed the PPIs to be almost fully optimized, with most single mutations giving a loss of affinity. In contrast, the non-optimized PPI for N-TIMP2/MMP-14 was reflected in a wide range of binding affinities, where single mutations exhibited a far more attenuated effect on the PPI. Our new platform reliably and comprehensively identified not only hot- and cold-spot residues, but also specificity-switch mutations that shape target affinity and specificity. Thus, our approach provides a methodology giving an unprecedentedly rich quantitative analysis of the binding specificity landscape, which will broaden the understanding of the mechanisms and evolutionary origins of specific PPIs and facilitate the rational design of specific inhibitors for structurally similar target proteins.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

Cited by 4 articles. 订阅此论文施引文献 订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3