Isomer-Specific Interactions of X-Pro Motifs: Investigating Fundamental Mechanisms of Signaling by Pro-Rich Sequences

这个项目提出了一种新的假说，即细胞内特定的“开/关”开关是如何被调节的。感兴趣的开关涉及两个特定蛋白质之间的生物信号相互作用，这两个蛋白质将先天免疫信号(细胞的第一道防线)与细胞迁移(细胞的定向移动)联系起来。这项工作建立在实验观察的基础上，即IRAK1(先天性免疫中的关键信号蛋白)采用两种非常不同且缓慢的相互转换结构(特定Trp-Pro肽键的顺式和反式异构体)，恰好位于它与Vasp(一种调节细胞迁移的蛋白质)结合的区域。VASP有两个不同的域(EVH1和EVH2)。只有VASP-EVH1结构域直接与IRAK1结合，并且这种相互作用只对IRAK1的反式异构体具有选择性。由于在Vasp进入图像之前，IRAK1具有几乎相等的顺式和反式结构群体，因此最初的IRAK1：VASP结合仅利用现有总IRAK1的大约一半。这个快速的初始结合步骤之后是一个缓慢的结合阶段，其速度取决于顺式和反式异构体的来回翻转的速度。这种缓慢的速度可以被催化翻转过程的一种酶(亲环素A，或CypA)大大加快。因此，CypA通过促进顺式到反式的快速转化来取代与Vasp结合的反式，从而有效地催化IRAK1-Vasp的结合作用。Vasp的EVH2结构域自结合为四个簇(四聚体)；因此，全长Vasp是一个四聚体，它显示四个EVH1结构域非常接近。有趣的是，最近的发现表明，当先天免疫信号被触发时，激活的IRAK1也是一个四聚体。当置于四聚体：四聚体相互作用(开关的“开”状态)的背景下时，IRAK1四聚体中所有4个IRAK1组分都处于VASP结合能力反式状态的要少得多。在这种情况下，酶催化的顺式/反式翻转(异构化)速率的加速对于实现二元式“开/关”开关更为关键，在这种开关中，打开或关闭开关的时间常数足够短，以在细胞内部工作的快节奏动力学中有效。该项目将提供1)单体：IRAK1：VASP相互作用的动力学和热力学描述，以及2)定量分析与IRAK1的特定簇相关的结合增强，以及顺反异构化在生物相关时间尺度上实现这种增强的作用。综上所述，这些结果将揭示一个关键的生物信号相互作用是如何快速将先天性免疫信号与细胞迁移联系起来的。这项工作协同耦合到细胞生物学家，他们研究IRAK1和Vasp在生物过程中的参与，如先天免疫和细胞迁移，以及系统生物学家，他们对信号通路进行数学建模，并在这些通路中的关键步骤需要热力学和动力学数据。本项目的研究将以多种方式整合到一门大型(70-80名学生)研究生级别3学分的蛋白质结构、功能和动力学课程(BioMG 6310)的教学中。作为这门课的一部分，我们布置了一个基于团队的项目，让学生写一份使用蛋白质来“拯救世界”的提案(重点关注能源和环境)，并在学期末的公共海报会议上展示项目。这项活动促进了与康奈尔大学阿特金森可持续未来中心(ACSF)的联系，包括向不同的参观者展示ACSF的海报，以及任命PI为ACSF院士。这项由美国国家科学基金会赞助的研究将被纳入到BioMG 6310讲座中，以阐明基本概念。对代表不足的群体的接触包括为学习网(一家当地的青年发展机构，为农村和城市的多样化儿童提供服务)进行实验室参观，以及在与极客(从事同理心、知识和服务的毕业生)的年度重建旅行中，为新奥尔良的第7和第9区带来对科学的兴奋，极客是康奈尔的一个研究生组织，PI为其提供建议。PI在她的实验室担任本科生(独立学习和暑期研究)和研究生的研究导师。学生接受跨学科的技术培训，包括核磁共振、生物物理方法、数学建模、生物化学和细胞生物学。

This project addresses a novel hypothesis regarding how a particular "on/off" switch inside a cell is regulated. The switch of interest involves a biological signaling interaction between two specific proteins that connect innate immunity signaling (a cell's first line of defense) to cell migration (the directed movement of a cell). This work builds on the experimental observation that IRAK1 (a key signaling protein in innate immunity) adopts two very different and slowly interconverting structures (cis and trans isomers of a specific Trp-Pro peptide bond), exactly in the region where it binds to VASP (a protein that regulates cell migration). VASP has two distinct domains (EVH1 and EVH2). Only the VASP-EVH1 domain directly binds to IRAK1, and this interaction is selective for only the trans isomer of IRAK1. Since IRAK1 has nearly equal populations of cis and trans structures before VASP enters the picture, the initial IRAK1:VASP binding utilizes only about half of the total IRAK1 that is present. This fast initial binding step is followed by a slow binding phase, the rate of which depends on the rate of flipping back and forth of the cis and trans isomers. This slow rate can be greatly accelerated by an enzyme (cyclophilin A, or CypA) that catalyzes the flipping process. Hence, CypA effectively catalyzes the IRAK1-VASP binding interaction by facilitating rapid conversion of cis to trans to replace the trans that has bound to VASP. The EVH2 domain of VASP self-associates as a cluster of four (a tetramer); consequently, full-length VASP is a tetramer that displays four EVH1 domains in close proximity. Interestingly, recent discoveries suggest that when innate immunity signaling is triggered, activated IRAK1 is also a tetramer. When placed into the context of a tetramer:tetramer interaction (the "on" state of the switch), there is far less IRAK1 tetramer in which all 4 IRAK1 components are in the VASP-binding competent trans state. In this case, enzyme-catalyzed acceleration of the cis/trans flipping (isomerization) rate is even more critical for achieving a binary "on/off" switch, in which the time constants for turning the switch on or off are short enough to be effective in the fast paced dynamics of the inner workings of a cell. This project will provide 1) a kinetic and thermodynamic description of the monomer:monomer IRAK1:VASP interaction, and 2) a quantitative analysis of the binding enhancement associated with the specific clustering of IRAK1 and the role of cis-trans isomerization in achieving this enhancement on a biologically relevant timescale. Together, these results will reveal how a key biological signaling interaction works to rapidly connect innate immunity signaling to cell migration. This work synergistically couples to cell biologists who study the participation of IRAK1 and VASP in biological processes such as innate immunity and cell migration, and to systems biologists who mathematically model signaling pathways and who require thermodynamic and kinetic data for critical steps in such pathways.The research in this project will be integrated in several ways into the teaching of a large (70-80 students) graduate level 3-credit course on Protein Structure, Function and Dynamics (BioMG 6310). As part of this class, a team-based project is assigned in which students write a proposal to "save the world" using proteins (with focus on energy and the environment), and projects are presented at the end of the semester at a public poster session. This activity has fostered connections with Cornell's Atkinson Center for a Sustainable Future (ACSF), including display of the posters at ACSF to a diverse audience of visitors, and the appointment of the PI as an ACSF Faculty Fellow. This NSF-sponsored research will be incorporated into BioMG 6310 lectures to illustrate fundamental concepts. Outreach to underrepresented groups includes giving lab tours for The Learning Web (a local youth development agency that serves a diversity of rural and city children), and bringing excitement for science to the 7th and 9th Wards of New Orleans during annual rebuilding trips with GEEKS (Graduates Employing Empathy, Knowledge and Service), a Cornell graduate student organization that the PI advises. The PI serves as research mentor for undergraduate (independent study and summer research) and graduate students in her lab. Students are trained in an interdisciplinary set of techniques that include NMR, biophysical methods, mathematical modeling, biochemistry, and cell biology.