Functional Classification and Interaction Selectivity Landscape of the Human SH3 Domain Superfamily.

SRC homology 3 (SH3) domains are critical interaction modules that orchestrate the assembly of protein complexes involved in diverse biological processes. They facilitate transient protein-protein interactions by selectively interacting with proline-rich motifs (PRMs). A database search revealed 298 SH3 domains in 221 human proteins. Multiple sequence alignment of human SH3 domains is useful for phylogenetic analysis and determination of their selectivity towards PRM-containing peptides (PRPs). However, a more precise functional classification of SH3 domains is achieved by constructing a phylogenetic tree only from PRM-binding residues and using existing SH3 domain-PRP structures and biochemical data to determine the specificity within each of the 10 families for particular PRPs. In addition, the C-terminal proline-rich domain of the RAS activator SOS1 covers 13 of the 14 recognized proline-rich consensus sequence motifs, encompassing differential PRP pattern selectivity among all SH3 families. To evaluate the binding capabilities and affinities, we conducted fluorescence dot blot and polarization experiments using 25 representative SH3 domains and various PRPs derived from SOS1. Our analysis has identified 45 interacting pairs, with binding affinities ranging from 0.2 to 125 micromolar, out of 300 tested and potential new SH3 domain-SOS1 interactions. Furthermore, it establishes a framework to bridge the gap between SH3 and PRP interactions and provides predictive insights into the potential interactions of SH3 domains with PRMs based on sequence specifications. This novel framework has the potential to enhance the understanding of protein networks mediated by SH3 domain-PRM interactions and be utilized as a general approach for other domain-peptide interactions.

A high-confidence Physcomitrium patens plasmodesmata proteome by iterative scoring and validation reveals diversification of cell wall proteins during evolution.

Plasmodesmata (PD) facilitate movement of molecules between plant cells. Regulation of this movement is still not understood. Plasmodesmata are hard to study, being deeply embedded within cell walls and incorporating several membrane types. Thus, structure and protein composition of PD remain enigmatic. Previous studies of PD protein composition identified protein lists with few validations, making functional conclusions difficult. We developed a PD scoring approach in iteration with large-scale systematic localization, defining a high-confidence PD proteome of Physcomitrium patens (HC300). HC300, together with bona fide PD proteins from literature, were placed in Pddb. About 65% of proteins in HC300 were not previously PD-localized. Callose-degrading glycolyl hydrolase family 17 (GHL17) is an abundant protein family with representatives across evolutionary scale. Among GHL17s, we exclusively found members of one phylogenetic clade with PD localization and orthologs occur only in species with developed PD. Phylogenetic comparison was expanded to xyloglucan endotransglucosylases/hydrolases and Exordium-like proteins, which also diversified into PD-localized and non-PD-localized members on distinct phylogenetic clades. Our high-confidence PD proteome HC300 provides insights into diversification of large protein families. Iterative and systematic large-scale localization across plant species strengthens the reliability of HC300 as basis for exploring structure, function, and evolution of this important organelle.

CDC42-IQGAP Interactions Scrutinized: New Insights into the Binding Properties of the GAP-Related Domain.

The IQ motif-containing GTPase-activating protein (IQGAP) family composes of three highly-related and evolutionarily conserved paralogs (IQGAP1, IQGAP2 and IQGAP3), which fine tune as scaffolding proteins numerous fundamental cellular processes. IQGAP1 is described as an effector of CDC42, although its effector function yet re-mains unclear. Biophysical, biochemical and molecular dynamic simulation studies have proposed that IQGAP RASGAP-related domains (GRDs) bind to the switch regions and the insert helix of CDC42 in a GTP-dependent manner. Our kinetic and equilibrium studies have shown that IQGAP1 GRD binds, in contrast to its C-terminal 794 amino acids (called C794), CDC42 in a nucleotide-independent manner indicating a binding outside the switch regions. To resolve this discrepancy and move beyond the one-sided view of GRD, we carried out affinity measurements and a systematic mutational analysis of the interfacing residues between GRD and CDC42 based on the crystal structure of the IQGAP2 GRD-CDC42Q61L GTP complex. We determined a 100-fold lower affinity of the GRD1 of IQGAP1 and of GRD2 of IQGAP2 for CDC42 mGppNHp in comparison to C794/C795 proteins. Moreover, partial and major mutation of CDC42 switch regions substantially affected C794/C795 binding but only a little GRD1 and remarkably not at all the GRD2 binding. However, we clearly showed that GRD2 contributes to the overall affinity of C795 by using a 11 amino acid mutated GRD variant. Furthermore, the GRD1 binding to the CDC42 was abolished using specific point mutations within the insert helix of CDC42 clearly supporting the notion that CDC42 binding site(s) of IQGAP GRD lies outside the switch regions among others in the insert helix. Collectively, this study provides further evidence for a mechanistic framework model that is based on a multi-step binding process, in which IQGAP GRD might act as a 'scaffolding domain' by binding CDC42 irrespective of its nucleotide-bound forms, followed by other IQGAP domains downstream of GRD that act as an effector domain and is in charge for a GTP-dependent interaction with CDC42.

Allosteric regulation of GRB2 modulates RAS activation.

RAS activation is a multiple-step process in which linkage of the extracellular stimuli to the RAS activator SOS1 is the main step in RAS activation. GRB2 adaptor protein is the main modulator in SOS1 recruitment to the plasma membrane and its activation. This interaction is well studied but the exact mechanism of GRB2-SOS1 complex formation and SOS1 activation has yet remained obscure. Here, a new allosteric mechanism for the GRB2 regulation is described as a prerequisite for the modulation of SOS1 activation. This regulatory mechanism comprises a series of intramolecular interactions which are potentiated by GRB2 interaction with upstream ligands.Abbreviations: GRB2, growth factor receptor-bound protein 2; SOS1, son of sevenless 1; RAS, Rat Sarcoma; GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; HER2, human epidermal growth factor receptor; SH3, SRC Homology 3; SH2, SRC Homology 2; PRD, proline-rich domain; PRM, proline-rich motif; PRP, proline-rich peptide; RTK, receptor tyrosine kinases.

The Pseudo-Natural Product Rhonin Targets RHOGDI.

For the discovery of novel chemical matter generally endowed with bioactivity, strategies may be particularly efficient that combine previous insight about biological relevance, e.g., natural product (NP) structure, with methods that enable efficient coverage of chemical space, such as fragment-based design. We describe the de novo combination of different 5-membered NP-derived N-heteroatom fragments to structurally unprecedented "pseudo-natural products" in an efficient complexity-generating and enantioselective one-pot synthesis sequence. The pseudo-NPs inherit characteristic elements of NP structure but occupy areas of chemical space not covered by NP-derived chemotypes, and may have novel biological targets. Investigation of the pseudo-NPs in unbiased phenotypic assays and target identification led to the discovery of the first small-molecule ligand of the RHO GDP-dissociation inhibitor 1 (RHOGDI1), termed Rhonin. Rhonin inhibits the binding of the RHOGDI1 chaperone to GDP-bound RHO GTPases and alters the subcellular localization of RHO GTPases.

Electrostatic Forces Mediate the Specificity of RHO GTPase-GDI Interactions.

Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylgeranylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.

The intramolecular allostery of GRB2 governing its interaction with SOS1 is modulated by phosphotyrosine ligands.

Growth factor receptor-bound protein 2 (GRB2) is a trivalent adaptor protein and a key element in signal transduction. It interacts via its flanking nSH3 and cSH3 domains with the proline-rich domain (PRD) of the RAS activator SOS1 and via its central SH2 domain with phosphorylated tyrosine residues of receptor tyrosine kinases (RTKs; e.g. HER2). The elucidation of structural organization and mechanistic insights into GRB2 interactions, however, remain challenging due to their inherent flexibility. This study represents an important advance in our mechanistic understanding of how GRB2 links RTKs to SOS1. Accordingly, it can be proposed that (1) HER2 pYP-bound SH2 potentiates GRB2 SH3 domain interactions with SOS1 (an allosteric mechanism); (2) the SH2 domain blocks cSH3, enabling nSH3 to bind SOS1 first before cSH3 follows (an avidity-based mechanism); and (3) the allosteric behavior of cSH3 to other domains appears to be unidirectional, although there is an allosteric effect between the SH2 and SH3 domains.

Accessory proteins of the RAS-MAPK pathway: moving from the side line to the front line.

Health and disease are directly related to the RTK-RAS-MAPK signalling cascade. After more than three decades of intensive research, understanding its spatiotemporal features is afflicted with major conceptual shortcomings. Here we consider how the compilation of a vast array of accessory proteins may resolve some parts of the puzzles in this field, as they safeguard the strength, efficiency and specificity of signal transduction. Targeting such modulators, rather than the constituent components of the RTK-RAS-MAPK signalling cascade may attenuate rather than inhibit disease-relevant signalling pathways.

A comprehensive analysis of RAS-effector interactions reveals interaction hotspots and new binding partners.

RAS effectors specifically interact with GTP-bound RAS proteins to link extracellular signals to downstream signaling pathways. These interactions rely on two types of domains, called RAS-binding (RB) and RAS association (RA) domains, which share common structural characteristics. Although the molecular nature of RAS-effector interactions is well-studied for some proteins, most of the RA/RB-domain-containing proteins remain largely uncharacterized. Here, we searched through human proteome databases, extracting 41 RA domains in 39 proteins and 16 RB domains in 14 proteins, each of which can specifically select at least one of the 25 members in the RAS family. We next comprehensively investigated the sequence-structure-function relationship between different representatives of the RAS family, including HRAS, RRAS, RALA, RAP1B, RAP2A, RHEB1, and RIT1, with all members of RA domain family proteins (RASSFs) and the RB-domain-containing CRAF. The binding affinity for RAS-effector interactions, determined using fluorescence polarization, broadly ranged between high (0.3 µM) and very low (500 µM) affinities, raising interesting questions about the consequence of these variable binding affinities in the regulation of signaling events. Sequence and structural alignments pointed to two interaction hotspots in the RA/RB domains, consisting of an average of 19 RAS-binding residues. Moreover, we found novel interactions between RRAS1, RIT1, and RALA and RASSF7, RASSF9, and RASSF1, respectively, which were systematically explored in sequence-structure-property relationship analysis, and validated by mutational analysis. These data provide a set of distinct functional properties and putative biological roles that should now be investigated in the cellular context.

Novel FMRP interaction networks linked to cellular stress.

Silencing of the fragile X mental retardation 1 (FMR1) gene and consequently lack of synthesis of FMR protein (FMRP) are associated with fragile X syndrome, which is one of the most prevalent inherited intellectual disabilities, with additional roles in increased viral infection, liver disease, and reduced cancer risk. FMRP plays critical roles in chromatin dynamics, RNA binding, mRNA transport, and mRNA translation. However, the underlying molecular mechanisms, including the (sub)cellular FMRP protein networks, remain elusive. Here, we employed affinity pull-down and quantitative LC-MS/MS analyses with FMRP. We identified known and novel candidate FMRP-binding proteins as well as protein complexes. FMRP interacted with 180 proteins, 28 of which interacted with its N terminus. Interaction with the C terminus of FMRP was observed for 102 proteins, and 48 proteins interacted with both termini. This FMRP interactome comprises known FMRP-binding proteins, including the ribosomal proteins FXR1P, NUFIP2, Caprin-1, and numerous novel FMRP candidate interacting proteins that localize to different subcellular compartments, including CARF, LARP1, LEO1, NOG2, G3BP1, NONO, NPM1, SKIP, SND1, SQSTM1, and TRIM28. Our data considerably expand the protein and RNA interaction networks of FMRP, which thereby suggest that, in addition to its known functions, FMRP participates in transcription, RNA metabolism, ribonucleoprotein stress granule formation, translation, DNA damage response, chromatin dynamics, cell cycle regulation, ribosome biogenesis, miRNA biogenesis, and mitochondrial organization. Thus, FMRP seems associated with multiple cellular processes both under normal and cell stress conditions in neuronal as well as non-neuronal cell types, as exemplified by its role in the formation of stress granules.

Activating Mutations of RRAS2 Are a Rare Cause of Noonan Syndrome.

Aberrant signaling through pathways controlling cell response to extracellular stimuli constitutes a central theme in disorders affecting development. Signaling through RAS and the MAPK cascade controls a variety of cell decisions in response to cytokines, hormones, and growth factors, and its upregulation causes Noonan syndrome (NS), a developmental disorder whose major features include a distinctive facies, a wide spectrum of cardiac defects, short stature, variable cognitive impairment, and predisposition to malignancies. NS is genetically heterogeneous, and mutations in more than ten genes have been reported to underlie this disorder. Despite the large number of genes implicated, about 10%-20% of affected individuals with a clinical diagnosis of NS do not have mutations in known RASopathy-associated genes, indicating that additional unidentified genes contribute to the disease, when mutated. By using a mixed strategy of functional candidacy and exome sequencing, we identify RRAS2 as a gene implicated in NS in six unrelated subjects/families. We show that the NS-causing RRAS2 variants affect highly conserved residues localized around the nucleotide binding pocket of the GTPase and are predicted to variably affect diverse aspects of RRAS2 biochemical behavior, including nucleotide binding, GTP hydrolysis, and interaction with effectors. Additionally, all pathogenic variants increase activation of the MAPK cascade and variably impact cell morphology and cytoskeletal rearrangement. Finally, we provide a characterization of the clinical phenotype associated with RRAS2 mutations.