Integrating disorder in globular multidomain proteins: Fuzzy sensors and the role of SH3 domains.

Intrinsically disordered proteins represent about one third of eukaryotic proteins. An additional third correspond to proteins containing folded domains as well as large intrinsically disordered regions (IDR). While IDRs may represent functionally autonomous domains, in some instances it has become clear that they provide a new layer of regulation for the activity displayed by the folded domains. The sensitivity of the conformational ensembles defining the properties of IDR to small changes in the cellular environment and the capacity to modulate this response through post-translational modifications makes IDR ideal sensors enabling continuous, integrative responses to complex cellular inputs. Folded domains (FD), on the other hand, are ideal effectors, e.g. by catalyzing enzymatic reactions or participating in binary on/off switches. In this perspective review we discuss the possible role of intramolecular fuzzy complexes to integrate the very different dynamic scales of IDR and FD, inspired on the recent observations of such dynamic complexes in Src family kinases, and we explore the possible general role of the SH3 domains connecting IDRs and FD.

Farseer-NMR: automatic treatment, analysis and plotting of large, multi-variable NMR data.

We present Farseer-NMR ( https://git.io/vAueU ), a software package to treat, evaluate and combine NMR spectroscopic data from sets of protein-derived peaklists covering a range of experimental conditions. The combined advances in NMR and molecular biology enable the study of complex biomolecular systems such as flexible proteins or large multibody complexes, which display a strong and functionally relevant response to their environmental conditions, e.g. the presence of ligands, site-directed mutations, post translational modifications, molecular crowders or the chemical composition of the solution. These advances have created a growing need to analyse those systems' responses to multiple variables. The combined analysis of NMR peaklists from large and multivariable datasets has become a new bottleneck in the NMR analysis pipeline, whereby information-rich NMR-derived parameters have to be manually generated, which can be tedious, repetitive and prone to human error, or even unfeasible for very large datasets. There is a persistent gap in the development and distribution of software focused on peaklist treatment, analysis and representation, and specifically able to handle large multivariable datasets, which are becoming more commonplace. In this regard, Farseer-NMR aims to close this longstanding gap in the automated NMR user pipeline and, altogether, reduce the time burden of analysis of large sets of peaklists from days/weeks to seconds/minutes. We have implemented some of the most common, as well as new, routines for calculation of NMR parameters and several publication-quality plotting templates to improve NMR data representation. Farseer-NMR has been written entirely in Python and its modular code base enables facile extension.

The pathogen-encoded signalling receptor Tir exploits host-like intrinsic disorder for infection.

The translocated intimin receptor (Tir) is an essential type III secretion system (T3SS) effector of attaching and effacing pathogens contributing to the global foodborne disease burden. Tir acts as a cell-surface receptor in host cells, rewiring intracellular processes by targeting multiple host proteins. We investigated the molecular basis for Tir's binding diversity in signalling, finding that Tir is a disordered protein with host-like binding motifs. Unexpectedly, also are several other T3SS effectors. By an integrative approach, we reveal that Tir dimerises via an antiparallel OB-fold within a highly disordered N-terminal cytosolic domain. Also, it has a long disordered C-terminal cytosolic domain partially structured at host-like motifs that bind lipids. Membrane affinity depends on lipid composition and phosphorylation, highlighting a previously unrecognised host interaction impacting Tir-induced actin polymerisation and cell death. Furthermore, multi-site tyrosine phosphorylation enables Tir to engage host SH2 domains in a multivalent fuzzy complex, consistent with Tir's scaffolding role and binding promiscuity. Our findings provide insights into the intracellular Tir domains, highlighting the ability of T3SS effectors to exploit host-like protein disorder as a strategy for host evasion.

A Myristoyl-Binding Site in the SH3 Domain Modulates c-Src Membrane Anchoring.

The c-Src oncogene is anchored to the cytoplasmic membrane through its N-terminal myristoylated SH4 domain. This domain is part of an intramolecular fuzzy complex with the SH3 and Unique domains. Here we show that the N-terminal myristoyl group binds to the SH3 domain in the proximity of the RT loop, when Src is not anchored to a lipid membrane. Residues in the so-called Unique Lipid Binding Region modulate this interaction. In the presence of lipids, the myristoyl group is released from the SH3 domain and inserts into the lipid membrane. The fuzzy complex with the SH4 and Unique domains is retained in the membrane-bound form, placing the SH3 domain close to the membrane surface and restricting its orientation. The apparent affinity of myristoylated proteins containing the SH4, Unique, and SH3 domains is modulated by these intramolecular interactions, suggesting a mechanism linking c-Src activation and membrane anchoring.

Intramolecular Fuzzy Interactions Involving Intrinsically Disordered Domains.

Structural disorder is an essential ingredient for function in many proteins and protein complexes. Fuzzy complexes describe the many instances where disorder is maintained as a critical element of protein interactions. In this minireview we discuss how intramolecular fuzzy interactions function in signaling complexes. Focussing on the Src family of kinases, we argue that the intrinsically disordered domains that are unique for each of the family members and display a clear fingerprint of long range interactions in Src, might have critical roles as functional sensor or effectors and mediate allosteric communication via fuzzy interactions.

The Unique Domain Forms a Fuzzy Intramolecular Complex in Src Family Kinases.

The N-terminal regulatory region of c-Src including the SH4, Unique, and SH3 domains adopts a compact, yet highly dynamic, structure that can be described as an intramolecular fuzzy complex. Most of the long-range interactions within the Unique domain are also observed in constructs lacking the structured SH3, indicating a considerable degree of preorganization of the disordered Unique domain. Here we report that members of the Src family of kinases (SFK) share well-conserved sequence features involving aromatic residues in their Unique domains. This observation contrasts with the supposed lack of sequence homology implied by the name of these domains and suggests that the other members of SFK also have a regulatory region involving their Unique domains. We argue that the Unique domain of each SFK is sensitive to specific input signals, encoded by each specific sequence, but the entire family shares a common mechanism for connecting the disordered and structured domains.

The SH3 Domain Acts as a Scaffold for the N-Terminal Intrinsically Disordered Regions of c-Src.

Regulation of c-Src activity by the intrinsically disordered Unique domain has recently been demonstrated. However, its connection with the classical regulatory mechanisms is still missing. Here we show that the Unique domain is part of a long loop closed by the interaction of the SH4 and SH3 domains. The conformational freedom of the Unique domain is further restricted through direct contacts with SH3 that are allosterically modulated by binding of a poly-proline ligand in the presence and in the absence of lipids. Our results highlight the scaffolding role of the SH3 domain for the c-Src N-terminal intrinsically disordered regions and suggest a connection between the regulatory mechanisms involving the SH3 and Unique domains.