Mapping, modeling and manipulating the interactions of protein domains that bind short linear motifs

映射、建模和操纵结合短线性基序的蛋白质结构域的相互作用

• 批准号: 10242750
• 负责人: AMY E KEATING
• 金额: $ 32.67万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242750
• 关键词: Actins; Affect; Affinity; Benchmarking; Binding; Binding Proteins; Biochemical; Biological; Biology; Biophysics; Cancer Control; Cell surface; Cells; Cellular biology; Characteristics; Code; Complex; Computing Methodologies; Consensus; Coupled; Cytoskeleton; Data; Development; Disease; Disseminated Malignant Neoplasm; Epitopes; Family; Family member; Fiber; Filopodia; Genetic Translation; Geometry; Goals; Human; Lead; Libraries; Malignant Neoplasms; Measurement; Measures; Mediating; Membrane Proteins; Methods; Modeling; Modernization; Molecular Biology; Molecular Probes; Multiprotein Complexes; NMR Spectroscopy; Neoplasm Metastasis; Neurons; Peptide Library; Peptides; Play; Proline; Protein Binding Domain; Protein Family; Protein Isoforms; Proteins; Proteome; RNA Interference; Reading; Reagent; Regulation; Reporting; Research; Resistance; Resources; Role; Screening Result; Signal Pathway; Signal Transduction; Specificity; Structural Biologist; Structural Models; Structure; Surface; Techniques; Tertiary Protein Structure; Testing; Therapeutic; Variant; Work; X-Ray Crystallography; base; cancer cell; cancer invasiveness; cell motility; chemotherapy; chromatin modification; computerized tools; design; experimental study; flexibility; human disease; inhibitor/antagonist; insight; model design; molecular recognition; neurodevelopment; paralogous gene; predictive modeling; predictive test; preference; programs; protein function; protein protein interaction; refractory cancer; scaffold; screening; therapeutic target; vasodilator-stimulated phosphoprotein

Many protein-protein interactions are mediated by short, linear motifs that bind selectively to modular, conserved protein domains. It is a long-standing goal of modern biology to understand the molecular recognition code for these types of interactions, because this would help elucidate mechanisms of signal transduction and predict cellular localization, targeting, and substrate selectivity of proteins that contain peptide-recognition domains. The ability to re-design domain-peptide interactions would lead to protein- interaction inhibitors useful for research and therapy. Recent advances in protein and peptide library screening and sequencing provide exciting opportunities to measure domain-peptide interactions in high throughput. In parallel with such measurements, computational methods are needed to interpret screening results and build models that can be used for protein interaction prediction and design. This proposal describes an integrated experimental/computational program for measuring, modeling and designing interactions mediated by structurally conserved EVH1 domains that bind to short, proline-rich motifs. The proposed studies focus on Ena/VASP family proteins critical for regulation of the actin cytoskeleton. Three human paralogs, Mena, VASP and EVL, act as scaffolds to assemble complexes at the ends of actin fibers. Ena/VASP proteins are critical for the formation of filopodia and lamellipodia and for regulation of actin- based cell motility; they also have newly discovered roles in neural development. Mena, and particularly its isoform Mena invasive, control cancer cell invasion and promote resistance to chemotherapy; the EVH1 binding activity of Mena is therefore an attractive therapeutic target. The peptide binding determinants for EVH1 domains are poorly understood. Short consensus binding motifs have been identified in earlier work but are not sufficient to predict which sequences will bind, or with what paralog specificity. Sequence that flanks known motifs modulates binding to EVH1 domains, but this phenomenon has not been systematically studied. In three specific aims, we propose to (1) experimentally identify Ena/VASP EVH1 domain-binding peptides in the human proteome, determine the sequence requirements for binding, and postulate new biologically relevant interaction partners, (2) apply computational structural modeling and experimental structure determination to understand the mechanisms of binding specificity, including the contributions of core-motif and flanking sequences, and (3) design high affinity and paralog- and/or isoform-selective inhibitors of EVH1 domain interactions. Deeper insight into how EVH1 domains engage their partners will advance our understanding of how these proteins contribute to cell motility, including in invasive cancer cells. Inhibitor design will provide reagents for perturbing Ena/VASP protein functions, including the role of Mena in regulating local mRNA translation in neurons. This work will establish a path for mapping and inhibiting domain-peptide interactions that can be applied to many other peptide-recognition domains.

许多蛋白质 - 蛋白质相互作用都是由短的线性基序介导的，这些基序有选择地与模块化结合 保守的蛋白质结构域。了解分子是现代生物学的长期目标 这些类型的互动的识别代码，因为这将有助于阐明信号的机制 转导和预测包含蛋白质的细胞定位，靶向和底物选择性 肽识别域。重新设计域肽相互作用的能力将导致蛋白质 - 相互作用抑制剂可用于研究和治疗。蛋白质和肽文库的最新进展 筛选和测序提供了令人兴奋的机会，以测量高的域肽相互作用 吞吐量。与此类测量并行，需要计算方法来解释筛选 结果和构建模型可用于蛋白质相互作用预测和设计。这个建议 描述用于测量，建模和设计的集成实验/计算程序 由结构保守的EVH1结构域介导的相互作用，这些结构域与短，富含脯氨酸的基序结合。这 拟议的研究集中于ENA/VASP家族蛋白，对于调节肌动蛋白细胞骨架至关重要。三 人类旁系同源物，北非，VASP和EVL充当脚手架，在肌动蛋白纤维的末端组装复合物。 ENA/VASP蛋白对于形成丝状和层状脂蛋白和肌动蛋白的调节至关重要 基于细胞的运动；他们在神经发展中也有新发现的作用。北非，尤其是它 同工型MENA侵入性，控制癌细胞侵袭并促进对化学疗法的耐药性； EVH1 因此，MENA的结合活性是一个有吸引力的治疗靶标。肽结合决定因素 EVH1域知之甚少。在早期工作中已经确定了简短的共识结合基准 但不足以预测哪些序列将结合，或与旁系同源性特异性结合。序列 侧面已知的基序调节与EVH1域结合，但是这种现象尚未系统地结合 研究。在三个特定目标中，我们建议（1）实验识别ENA/VASP EVH1域结合 人蛋白质组中的肽，确定结合的序列要求，并假设新的 生物学相关的相互作用伙伴，（2）应用计算结构建模和实验 结构确定以了解结合特异性的机制，包括 核心序列和侧翼序列，以及（3）设计高亲和力以及旁系同型和/或同工型选择性 EVH1域相互作用的抑制剂。更深入了解EVH1领域如何参与其合作伙伴将会 促进我们对这些蛋白质如何对细胞运动有何贡献的理解，包括在侵入性癌症中 细胞。抑制剂设计将为扰动ENA/VASP蛋白功能提供试剂，包括 MENA调节神经元中局部mRNA翻译。这项工作将为映射和 可以应用于许多其他肽识别域的抑制域肽相互作用。