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Dengue virus, particularly serotype 2 (DENV-2), poses a significant global health threat, and understanding the molecular basis of its interactions with host cell proteins is imperative for developing targeted therapeutic strategies. This study elucidated the interactions between proline-enriched motifs and Src homology 3 (SH3) domain. The SH3 domain is pivotal in mediating protein–protein interactions, particularly by recognizing and binding to proline-rich regions in partner proteins. Through a computational pipeline, we analyzed the interactions and binding modes of proline-enriched motifs with SH3 domains, identified new potential DENV-2 interactions with the SH3 domain, and revealed potential hot spot residues, underscoring their significance in the viral life cycle. This comprehensive analysis provides crucial insights into the molecular basis of DENV-2 infection, highlighting conserved and serotype-specific interactions. The identified hot spot residues offer potential targets for therapeutic intervention, laying the foundation for developing antiviral strategies against Dengue virus infection. These findings contribute to the broader understanding of viral–host interactions and provide a roadmap for future research on Dengue virus pathogenesis and treatment. [ABSTRACT FROM AUTHOR]

Growth-factor-receptor-binding protein 2 (GRB2) is a non-enzymatic adaptor protein that plays a pivotal role in precisely regulated signaling cascades from cell surface receptors to cellular responses, including signaling transduction and gene expression. GRB2 binds to numerous target molecules, thereby modulating a complex cell signaling network with diverse functions. The structural characteristics of GRB2 are essential for its functionality, as its multiple domains and interaction mechanisms underpin its role in cellular biology. The typical signaling pathway involving GRB2 is initiated by the ligand stimulation to its receptor tyrosine kinases (RTKs). The activation of RTKs leads to the recruitment of GRB2 through its SH2 domain to the phosphorylated tyrosine residues on the receptor. GRB2, in turn, binds to the Son of Sevenless (SOS) protein through its SH3 domain. This binding facilitates the activation of Ras, a small GTPase, which triggers a cascade of downstream signaling events, ultimately leading to cell proliferation, survival, and differentiation. Further research and exploration into the structure and function of GRB2 hold great potential for providing novel insights and strategies to enhance medical approaches for related diseases. In this review, we provide an outline of the proteins that engage with domains of GRB2, along with the function of different GRB2 domains in governing cellular signaling pathways. This furnishes essential points of current studies for the forthcoming advancement of therapeutic medications aimed at GRB2. [ABSTRACT FROM AUTHOR]

Truncation of the protein-protein interaction SH3 domain of the membrane remodeling Bridging Integrator 1 (BIN1, Amphiphysin 2) protein leads to centronuclear myopathy. Here, we assessed the impact of a set of naturally observed, previously uncharacterized BIN1 SH3 domain variants using conventional in vitro and cell-based assays monitoring the BIN1 interaction with dynamin 2 (DNM2) and identified potentially harmful ones that can be also tentatively connected to neuromuscular disorders. However, SH3 domains are typically promiscuous and it is expected that other, so far unknown partners of BIN1 exist besides DNM2, that also participate in the development of centronuclear myopathy. In order to shed light on these other relevant interaction partners and to get a holistic picture of the pathomechanism behind BIN1 SH3 domain variants, we used affinity interactomics. We identified hundreds of new BIN1 interaction partners proteome-wide, among which many appear to participate in cell division, suggesting a critical role of BIN1 in the regulation of mitosis. Finally, we show that the identified BIN1 mutations indeed cause proteome-wide affinity perturbation, signifying the importance of employing unbiased affinity interactomic approaches.

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. [ABSTRACT FROM AUTHOR]

The Bin–Amphiphysin–Rvs (BAR) domain of endophilin binds to the cell membrane and shapes it into a tubular shape for endocytosis. Endophilin has a Src-homology 3 (SH3) domain at their C-terminal. The SH3 domain interacts with the proline-rich motif (PRM) that is found in proteins such as neural Wiskott–Aldrich syndrome protein (N-WASP). Here, we re-examined the binding sites of the SH3 domain of endophilin in N-WASP by machine learning-based prediction and identified the previously unrecognized binding site. In addition to the well-recognized PRM at the central proline-rich region, we found a PRM in front of the N-terminal WASP homology 1 (WH1) domain of N-WASP (NtPRM) as a binding site of the endophilin SH3 domain. Furthermore, the diameter of the membrane tubules in the presence of NtPRM mutant was narrower and wider than that in the presence of N-WASP and in its absence, respectively. Importantly, the NtPRM of N-WASP was involved in the membrane localization of endophilin A2 in cells. Therefore, the NtPRM contributes to the binding of endophilin to N-WASP in membrane remodeling. [ABSTRACT FROM AUTHOR]

Human nonreceptor tyrosine kinase (TK) Fyn is implicated in various cellular processes and has been exploited as a sophisticated druggable target of pediatric T-cell lymphoma by blocking its kinase domain with small-molecule inhibitors or disrupting its regulatory SH2 and SH3 domains with peptidic inhibitors. In this study, the proline-heavy 9P1Y-peptide was found as a good binder of Fyn SH3 domain, of which the chemical diversity space was extended by replacing its proline residues with a variety of N-substituted amino acids, since N-substitution can mimic the side-chain location and shape of proline. A systematic single-point N-substituting perturbation profile (SSNPP) for 9P1Y-peptide was created theoretically, from which a systematic combinatorial peptoid library (SCPL) was then generated by introducing favorable N-substituted to different proline residue positions of the peptide. The computational design was further substantiated by fluorescence spectroscopy assays to identify five promising peptoid hits in the SCPL as potential Fyn SH3 binders. In particular, the designed peptoid Ptoid2 exhibited a good affinity to Fyn SH3 domain ( K d = 0. 7 6 μ M) and a moderate selectivity for Fyn over Lyn (3.8-fold), which are improved substantially from the natural 9P1Y-peptide. Molecular mechanism underlying the N-substituting perturbation effect was also examined in detail. The chemical diversity space of a Fyn SH3 binder is exploited by replacing its proline residues with a variety of N-substituted amino acids. A systematic single-point N-substituting perturbation profile is created and a systematic combinatorial peptoid library is generated by introducing favorable N-substituted to different proline residues. [ABSTRACT FROM AUTHOR]

Drk, a homologue of human GRB2 in Drosophila, receives signals from outside the cells through the interaction of its SH2 domain with the phospho-tyrosine residues in the intracellular regions of receptor tyrosine kinases (RTKs) such as Sevenless, and transduces the signals downstream through the association of its N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) with proline-rich motifs (PRMs) in Son of Sevenless (Sos) or Daughter of Sevenless (Dos). Isolated Drk-NSH3 exhibits a conformational equilibrium between the folded and unfolded states, while Drk-CSH3 adopts only a folded confirmation. Drk interacts with PRMs of the PxxPxR motif in Sos and the PxxxRxxKP motif in Dos. Our previous study has shown that Drk-CSH3 can bind to Sos, but the interaction between Drk-NSH3 and Dos has not been investigated. To assess the affinities of both SH3 domains towards Sos and Dos, we conducted NMR titration experiments using peptides derived from Sos and Dos. Sos-S1 binds to Drk-NSH3 with the highest affinity, strongly suggesting that the Drk-Sos multivalent interaction is initiated by the binding of Sos-S1 and NSH3. Our results also revealed that the two Sos-derived PRMs clearly favour NSH3 for binding, whereas the two Dos-derived PRMs show almost similar affinity for NSH3 and CSH3. We have also performed docking simulations based on the chemical shift perturbations caused by the addition of Sos- and Dos-derived peptides. Finally, we discussed the various modes in the interactions of Drk with Sos/Dos. [ABSTRACT FROM AUTHOR]

SRC homology 3 (SH3) domains are fundamental modules that enable the assembly of protein complexes through physical interactions with a pool of proline-rich/noncanonical motifs from partner proteins. They are widely studied modular building blocks across all five kingdoms of life and viruses, mediating various biological processes. The SH3 domains are also implicated in the development of human diseases, such as cancer, leukemia, osteoporosis, Alzheimer's disease, and various infections. A database search of the human proteome reveals the existence of 298 SH3 domains in 221 SH3 domain-containing proteins (SH3DCPs), ranging from 13 to 720 kilodaltons. A phylogenetic analysis of human SH3DCPs based on their multi-domain architecture seems to be the most practical way to classify them functionally, with regard to various physiological pathways. This review further summarizes the achievements made in the classification of SH3 domain functions, their binding specificity, and their significance for various diseases when exploiting SH3 protein modular interactions as drug targets. [ABSTRACT FROM AUTHOR]

Charged residues on the surface of proteins are critical for both protein stability and interactions. However, many proteins contain binding regions with a high net charge that may destabilize the protein but are useful for binding to oppositely charged targets. We hypothesized that these domains would be marginally stable, as electrostatic repulsion would compete with favorable hydrophobic collapse during folding. Furthermore, by increasing the salt concentration, we predict that these protein folds would be stabilized by mimicking some of the favorable electrostatic interactions that take place during target binding. We varied the salt and urea concentrations to probe the contributions of electrostatic and hydrophobic interactions for the folding of the yeast SH3 domain found in Abp1p. The SH3 domain was significantly stabilized with increased salt concentrations due to Debye–Huckel screening and a nonspecific territorial ion‐binding effect. Molecular dynamics and NMR show that sodium ions interact with all 15 acidic residues but do little to change backbone dynamics or overall structure. Folding kinetics experiments show that the addition of urea or salt primarily affects the folding rate, indicating that almost all the hydrophobic collapse and electrostatic repulsion occur in the transition state. After the transition state formation, modest yet favorable short‐range salt bridges are formed along with hydrogen bonds, as the native state fully folds. Thus, hydrophobic collapse offsets electrostatic repulsion to ensure this highly charged binding domain can still fold and be ready to bind to its charged peptide targets, a property that is likely evolutionarily conserved over 1 billion years. [ABSTRACT FROM AUTHOR]

LYN proto-oncogene, Src family tyrosine kinase (Lyn) is a tyrosine kinase that belongs to the Src family (SFK). It is expressed as two isoforms in humans, LynA and LynB. Like other SFKs, Lyn consists of five protein domains, an N-terminal SH4 domain followed by a unique domain, the SH3 and SH2 domains, and a catalytic SH1 domain. The autophosphorylation of Tyr397 activates the protein, while the phosphorylation of the C-terminal inhibitory Tyr508 by C-terminal Src kinase (Csk) or Csk homologous kinase (Chk) inhibits the catalytic activity. The interaction of the SH2 domain with the phosphorylated Tyr508 stabilizes a compact, self-inhibited state. The interaction of the SH3 domain with a linker between the SH2 and catalytic domains further stabilizes this inactive conformation. The two critical structural features of the catalytic domain are a conserved DFG moiety and the αC helix, which can adopt in or out conformations. In the active state, both the DFG moiety and αC helix adopt in conformations, while in the inactive state, they adopt out conformations. Lyn has well-established functions in various hematopoietic cell types and more recent studies have revealed its roles in non-hematopoietic cells. At the molecular level, these functions are mainly exerted by phosphorylating specific tyrosine residues in immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activator motifs (ITAMs) associated with cell surface receptors. The phosphorylation of ITAMs by Lyn can initiate either activating or inhibitory (ITAMi) cell signaling depending on the receptor, targeting mode (crosslinking or monovalent targeting), and the cellular context. The phosphorylation of ITIMs by Lyn initiates inhibitory cell signaling via the recruitment of phosphatases to the ITIM-bearing receptor. The role of Lyn in cancer and autoimmune diseases has been extensively discussed in the literature. The involvement of Lyn in neurodegenerative diseases has been described more recently and, as such, it is now an emerging target for the treatment of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

The activation of Src kinase in cells is strictly controlled by intramolecular inhibitory interactions mediated by SH3 and SH2 domains. They impose structural constraints on the kinase domain holding it in a catalytically non-permissive state. The transition between inactive and active conformation is known to be largely regulated by the phosphorylation state of key tyrosines 416 and 527. Here, we identified that phosphorylation of tyrosine 90 reduces binding affinity of the SH3 domain to its interacting partners, opens the Src structure, and renders Src catalytically active. This is accompanied by an increased affinity to the plasma membrane, decreased membrane motility, and slower diffusion from focal adhesions. Phosphorylation of tyrosine 90 controlling SH3-medited intramolecular inhibitory interaction, analogical to tyrosine 527 regulating SH2-C-terminus bond, enables SH3 and SH2 domains to serve as cooperative but independent regulatory elements. This mechanism allows Src to adopt several distinct conformations of varying catalytic activities and interacting properties, enabling it to operate not as a simple switch but as a tunable regulator functioning as a signalling hub in a variety of cellular processes.

Growth-factor-receptor-binding protein 2 (GRB2) is a non-enzymatic adaptor protein that plays a pivotal role in precisely regulated signaling cascades from cell surface receptors to cellular responses, including signaling transduction and gene expression. GRB2 binds to numerous target molecules, thereby modulating a complex cell signaling network with diverse functions. The structural characteristics of GRB2 are essential for its functionality, as its multiple domains and interaction mechanisms underpin its role in cellular biology. The typical signaling pathway involving GRB2 is initiated by the ligand stimulation to its receptor tyrosine kinases (RTKs). The activation of RTKs leads to the recruitment of GRB2 through its SH2 domain to the phosphorylated tyrosine residues on the receptor. GRB2, in turn, binds to the Son of Sevenless (SOS) protein through its SH3 domain. This binding facilitates the activation of Ras, a small GTPase, which triggers a cascade of downstream signaling events, ultimately leading to cell proliferation, survival, and differentiation. Further research and exploration into the structure and function of GRB2 hold great potential for providing novel insights and strategies to enhance medical approaches for related diseases. In this review, we provide an outline of the proteins that engage with domains of GRB2, along with the function of different GRB2 domains in governing cellular signaling pathways. This furnishes essential points of current studies for the forthcoming advancement of therapeutic medications aimed at GRB2.

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.

Human osteoclast-stimulating factor (OSF) induces osteoclast formation and bone resorption in osteoporosis by recruiting multiple signaling complexes with downstream partners. Protein contains a peptide-recognition Src homology 3 (SH3) domain that can recognize and bind class II linear motif P − 1 x x P + 2 x [ K ∕ R ] + 4 to its partner proteins. The motif is defined by two prolines at positions − 1 and + 2, which are the primary anchor residues required for the domain recognition, and a positively charged amino acid at position + 4, which is the secondary anchor residue and determines the binding orientation of the motif peptides on the domain surface. In this study, we systematically examined the intermolecular interaction of OSF SH3 domain with a high-affinity decapeptide segment derived from its partner protein Sam68 at structural and energetic levels. It was found that, in addition to the primary and secondary anchor residues, the residue at peptide position + 1 is also important, which can form a π -stacking system (consisting of multiple cation- π or π – π stacking interactions) with its vicinal aromatic residues Phe23, Trp49 and Tyr65 of OSF SH3 domain, thus, largely stabilizing the domain–peptide complex. Here, we assigned the position + 1 as the third anchor residue and investigated the stacking effect by systematically substituting the position + 1 residue with six charged/aromatic amino acids (Arg, Lys, His, Phe, Tyr and Trp) and one neutral amino acid (Ala), as well as their impacts on the domain–peptide binding. A strong stacking effect was observed in association with charged/aromatic substitutions relative to neutral substitution, conferring substantial stability to the complex formation. A further fluorescence-based assay also substantiated the computational findings; the lysine and tyrosine substitutions ( K + 1 and Y + 1 ) were observed to significantly and moderately improve peptide affinity by 4.7-fold and 1.4-fold relative to wild-type Sam68 decapeptide ( R + 1 ), respectively. The binding behavior of peptide ligands to human OSF SH3 domain is systematically analyzed at structural and energetic levels. The peptide position +1 is involved in a π-stacking system with its vicinal aromatic domain residues. The peptide position +1 is assigned as third anchor residue. The π-stacking system is rationally designed by substituting the position +1 residue with diverse aromatic/charged amino acids. [ABSTRACT FROM AUTHOR]

RHOV and RHOU are considered atypical Rho-family small GTPases because of the existence of N- and C-terminal extension regions, abnormal GDP/GTP cycling, and post-translational modification. Particularly, RHOV and RHOU both have a proline-rich (PR) motif in the N-terminal region. It has been reported that the PR motif of RHOU interacts with GRB2, a SH3 domain-containing adaptor protein, and regulates its activity through EGF receptor signaling. However, it is unknown whether RHOV, like RHOU, interacts with SH3 domain-containing adaptor proteins. In this study, we investigated the interactions between RHOV and SH3 domain-containing adaptor proteins, including GRB2 and NCK2. The RHOV-induced serum response factor (SRF)-dependent gene transcriptional activity was attenuated in cells co-expressing either GRB2 or NCK2 compared to cells expressing RHOV alone. From the results of experiments using various gene mutants of RHOV and GRB2, it appears that the PR motif of the N-terminal region of RHOV is the crucial binding site for the SH3 domain-containing proteins. Furthermore, we found that Ser25 in the N-terminal region of RHOV is phosphorylated by PKA and that its phosphorylation is suppressed by interaction with NCK2 but not GRB2. We have found a novel regulatory mechanism for the phosphorylation of RHOV and its interaction with SH3 domain-containing adaptor proteins. [Display omitted] • GRB2 and NCK2 attenuated RHOV signaling through interaction between the SH3 domain and the proline-rich region of the RHOV N-terminal. • GRB2 suppresses RHOV-mediated-filopodia formation. • Phosphorylation of RHOV Ser25 by PKA is suppressed by interaction with NCK2 but not GRB2. [ABSTRACT FROM AUTHOR]

Drk, a homologue of human GRB2 in Drosophila, receives signals from outside the cells through the interaction of its SH2 domain with the phospho-tyrosine residues in the intracellular regions of receptor tyrosine kinases (RTKs) such as Sevenless, and transduces the signals downstream through the association of its N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) with proline-rich motifs (PRMs) in Son of Sevenless (Sos) or Daughter of Sevenless (Dos). Isolated Drk-NSH3 exhibits a conformational equilibrium between the folded and unfolded states, while Drk-CSH3 adopts only a folded confirmation. Drk interacts with PRMs of the PxxPxR motif in Sos and the PxxxRxxKP motif in Dos. Our previous study has shown that Drk-CSH3 can bind to Sos, but the interaction between Drk-NSH3 and Dos has not been investigated. To assess the affinities of both SH3 domains towards Sos and Dos, we conducted NMR titration experiments using peptides derived from Sos and Dos. Sos-S1 binds to Drk-NSH3 with the highest affinity, strongly suggesting that the Drk-Sos multivalent interaction is initiated by the binding of Sos-S1 and NSH3. Our results also revealed that the two Sos-derived PRMs clearly favour NSH3 for binding, whereas the two Dos-derived PRMs show almost similar affinity for NSH3 and CSH3. We have also performed docking simulations based on the chemical shift perturbations caused by the addition of Sos- and Dos-derived peptides. Finally, we discussed the various modes in the interactions of Drk with Sos/Dos.

Drk, a Drosophila homologue of human GRB2, interacts with Sevenless (Sev) receptor via its SH2 domain, while the N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) are responsible for the interaction with proline-rich motifs (PRMs) of Son of sevenless (Sos) or Daughter of Sevenless (Dos). Drk-NSH3 on its own has a conformational equilibrium between folded and unfolded states, and the folded state is stabilised by the association with a Sos-derived proline-rich peptide with PxxPxR motif. In contrast, Drk-CSH3 is supposed to bind PxxxRxxKP motifs in Dos. Aiming at clarifying the structural and functional differences between the two SH3 domains, we performed NMR studies of Drk-CSH3. The resulting solution structure and the 15N-relaxation data showed that Drk-CSH3 consists of a stable domain. Large chemical shift perturbation was commonly found around the RT loop and the hydrophobic patch, while there were also changes that occur characteristically for Sos- or Dos-derived peptides. Sos-derived two peptides with PxxPxR motif showed stronger affinity to Drk-CSH3, indicating that the Sos PRMs can bind both N- and C-SH3 domains. Dos-derived two peptides could also bind Drk-CSH3, but with much weaker affinity, suggesting a possibility that any cooperative binding of Dos-PRMs may strengthen the Drk-Dos interaction. The NMR studies as well as the docking simulations provide valuable insights into the biological and biophysical functions of two SH3 domains in Drk. • We determined the high-resolution solution structure of Drosophila Drk-CSH3. • Unlike Drk-NSH3 in a fold/unfold equilibrium, Drk-CSH3 consists of a stable domain. • Sos-derived peptides with PxxPxR motif showed stronger affinity to Drk-CSH3. • Dos-derived peptides with PxxxRxxKP motif showed much weaker affinity to Drk-CSH3. [ABSTRACT FROM AUTHOR]

ASAP1 and its paralog ASAP2 belong to a PI 4,5 P 2 -dependent Arf GTPase-activating protein (Arf-GAP) family capable of modulating membrane and cytoskeletal dynamics. ASAPs regulate cell adhesive structures such as invadosomes and focal adhesions during cell attachment and migration. Malfunctioning of ASAP1 has been implicated in the malignant phenotypes of various cancers. Here, we discovered that the SH3 domain of ASAP1 or ASAP2 specifically binds to a 12-residue, positively charged peptide fragment from the 440 kDa giant ankyrin-B, a neuronal axon specific scaffold protein. The high-resolution structure of the ASAP1-SH3 domain in complex with the gAnkB peptide revealed a noncanonical SH3-ligand binding mode with high affinity and specificity. Structural analysis of the complex readily uncovered a consensus ASAP1-SH3 binding motif, which allowed the discovery of a number of previously unknown binding partners of ASAP1-SH3 including Clasp1/Clasp2, ALS2, β-Pix, DAPK3, PHIP, and Limk1. Fittingly, these newly identified ASAP1 binding partners are primarily key modulators of the cytoskeletons. Finally, we designed a cell-penetrating, highly potent ASAP1 SH3 domain binding peptide with a K d ∼7 nM as a tool for studying the roles of ASAPs in different cellular processes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Biomolecular condensates are microdroplets that form inside cells and serve to selectively concentrate proteins, RNAs and other molecules for a variety of physiological functions, but can contribute to cancer, neurodegenerative diseases and viral infections. The formation of these condensates is driven by weak, transient interactions between molecules. These weak associations can operate at the level of whole protein domains, elements of secondary structure or even moieties composed of just a few atoms. Different types of condensates do not generally combine to form larger microdroplets, suggesting that each uses a distinct class of attractive interactions. Here, we address whether polyproline II (PPII) helices mediate condensate formation. By combining with PPII‐binding elements such as GYF, WW, profilin, SH3 or OCRE domains, PPII helices help form lipid rafts, nuclear speckles, P‐body‐like neuronal granules, enhancer complexes and other condensates. The number of PPII helical tracts or tandem PPII‐binding domains can strongly influence condensate stability. Many PPII helices have a low content of proline residues, which hinders their identification. Recently, we characterized the NMR spectral properties of a Gly‐rich, Pro‐poor protein composed of six PPII helices. Based on those results, we predicted that many Gly‐rich segments may form PPII helices and interact with PPII‐binding domains. This prediction is being tested and could join the palette of verified interactions contributing to biomolecular condensate formation.

SRC homology 3 (SH3) domains are fundamental modules that enable the assembly of protein complexes through physical interactions with a pool of proline-rich/noncanonical motifs from partner proteins. They are widely studied modular building blocks across all five kingdoms of life and viruses, mediating various biological processes. The SH3 domains are also implicated in the development of human diseases, such as cancer, leukemia, osteoporosis, Alzheimer’s disease, and various infections. A database search of the human proteome reveals the existence of 298 SH3 domains in 221 SH3 domain-containing proteins (SH3DCPs), ranging from 13 to 720 kilodaltons. A phylogenetic analysis of human SH3DCPs based on their multi-domain architecture seems to be the most practical way to classify them functionally, with regard to various physiological pathways. This review further summarizes the achievements made in the classification of SH3 domain functions, their binding specificity, and their significance for various diseases when exploiting SH3 protein modular interactions as drug targets.

The nonreceptor tyrosine kinase ITK is a key component of the T cell receptor (TCR) signaling pathway and is required for cytokine production by CD4+ T cells that have differentiated into Th2 cells. Structural and biochemical studies suggest that contacts between the SH2 and SH3 domains of ITK mediate intermolecular self-association, forming a structure that restrains ITK activity by interfering with interactions between ITK and other components of the TCR signaling pathway. Wild-type (WT) ITK and a panel of ITK mutants containing amino acid substitutions in the SH2 and SH3 domains were tested for self-association and for binding to the adaptor protein SLP76, a key ligand for the ITK SH2 domain. WT and ITK mutants were also expressed in Itk-deficient CD4+ T cells via retroviral-mediated gene delivery to analyze their ability to support TCR signaling and cytokine production by Th2 cells. Specific amino acid substitutions in the ITK SH2 or SH3 domains impaired self-association, with the greatest effects being seen when both intermolecular SH2-SH3 domain contacts were disrupted. Two of the SH2 domain substitutions tested reduced ITK self-association but had no effect on binding to SLP-76. When their function was analyzed in Th2 cells, ITK proteins with diminished self-association activity supported greater IL-4 production and calcium flux in response to TCR stimulation compared to WT ITK. Our findings indicate that intermolecular contacts between ITK molecules can restrain the amplitude of TCR signaling, suggesting ITK is a limiting factor for responses by CD4+ T cells. [ABSTRACT FROM AUTHOR]

Recent evidence has suggested a possible role for progesterone receptor (PR) in the progression of non-small cell lung cancer (NSCLC). However, little is known concerning roles of PR in NSCLC. PR contains a polyproline domain (PPD), which directly binds to the SH3 domain of signaling molecules. Because PPD-SH3 interactions are essential for EGFR signaling, we hypothesized that the presence of PR-PPD interfered with EGFR-mediated signaling and cell proliferation. We examined the role of PR-PPD in cell proliferation and signaling by stably expressing PR-B, or PR-B with disrupting mutations in the PPD (PR-BΔSH3), from a tetracycline-regulated promoter in A549 NSCLC cells. PR-B dose-dependently inhibited cell growth in the absence of ligand, and progestin (R5020) treatment further suppressed the growth. Treatment with RU486 abolished PR-B- and R5020-mediated inhibition of cell proliferation. Expression of PR-BΔSH3 and treatment with R5020 or RU486 had no effect on cell proliferation. Furthermore, PR-B expression but not PR-BΔSH3 expression reduced EGF-induced A549 proliferation and activation of ERK1/2, in the absence of ligand. Taken together, our data demonstrated the significance of PR extranuclear signaling through PPD interactions in EGFR-mediated proliferation and signaling in NSCLC.

The maintenance of genome stability requires the coordinated actions of multiple proteins and protein complexes, that are collectively known as genome guardians. Within this broadly defined family is a subset of proteins that contain oligonucleotide/oligosaccharide-binding folds (OB-fold). While OB-folds are widely associated with binding to single-stranded DNA this view is no longer an accurate depiction of how these domains are utilized. Instead, the core of the OB-fold is modified and adapted to facilitate binding to a variety of DNA substrates (both single- and double-stranded), phospholipids, and proteins, as well as enabling catalytic function to a multi-subunit complex. The flexibility accompanied by distinctive oligomerization states and quaternary structures enables OB-fold genome guardians to maintain the integrity of the genome via a myriad of complex and dynamic, protein-protein; protein-DNA, and protein-lipid interactions in both prokaryotes and eukaryotes.

Evolution-based deep generative models represent an exciting direction in understanding and designing proteins. An open question is whether such models can learn specialized functional constraints that control fitness in specific biological contexts. Here, we examine the ability of generative models to produce synthetic versions of Src-homology 3 (SH3) domains that mediate signaling in the Sho1 osmotic stress response pathway of yeast. We show that a variational autoencoder (VAE) model produces artificial sequences that experimentally recapitulate the function of natural SH3 domains. More generally, the model organizes all fungal SH3 domains such that locality in the model latent space (but not simply locality in sequence space) enriches the design of synthetic orthologs and exposes non-obvious amino acid constraints distributed near and far from the SH3 ligand-binding site. The ability of generative models to design ortholog-like functions in vivo opens new avenues for engineering protein function in specific cellular contexts and environments., Competing Interests: Declaration of interests R.R. and A.L.F. are co-founders and consultants of Evozyne, Inc. and co-authors of US Patent Application 17/642,582 and International Patent Application PCT/US2020/050466. A.L.F. is also co-author of US Patent Application 16/887,710, US Provisional Patent Applications 63/314,898, 63/479,378, and 63/521,617 and International Patent Application PCT/US2020/035206., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Truncation of the protein-protein interaction SH3 domain of the membrane remodeling Bridging Integrator 1 (BIN1, Amphiphysin 2) protein leads to centronuclear myopathy. Here, we assessed the impact of a set of naturally observed, previously uncharacterized BIN1 SH3 domain variants using conventional in vitro and cell-based assays monitoring the BIN1 interaction with dynamin 2 (DNM2) and identified potentially harmful ones that can be also tentatively connected to neuromuscular disorders. However, SH3 domains are typically promiscuous and it is expected that other, so far unknown partners of BIN1 exist besides DNM2, that also participate in the development of centronuclear myopathy. In order to shed light on these other relevant interaction partners and to get a holistic picture of the pathomechanism behind BIN1 SH3 domain variants, we used affinity interactomics. We identified hundreds of new BIN1 interaction partners proteome-wide, among which many appear to participate in cell division, suggesting a critical role of BIN1 in the regulation of mitosis. Finally, we show that the identified BIN1 mutations indeed cause proteome-wide affinity perturbation, signifying the importance of employing unbiased affinity interactomic approaches., Competing Interests: BZ, EE, BM, LN, MP, ZD, SO, GT, JL, GG No competing interests declared, (© 2024, Zambo et al.)

Biomolecular condensates are microdroplets that form inside cells and serve to selectively concentrate proteins, RNAs and other molecules for a variety of physiological functions, but can contribute to cancer, neurodegenerative diseases and viral infections. The formation of these condensates is driven by weak, transient interactions between molecules. These weak associations can operate at the level of whole protein domains, elements of secondary structure or even moieties composed of just a few atoms. Different types of condensates do not generally combine to form larger microdroplets, suggesting that each uses a distinct class of attractive interactions. Here, we address whether polyproline II (PPII) helices mediate condensate formation. By combining with PPII‐binding elements such as GYF, WW, profilin, SH3 or OCRE domains, PPII helices help form lipid rafts, nuclear speckles, P‐body‐like neuronal granules, enhancer complexes and other condensates. The number of PPII helical tracts or tandem PPII‐binding domains can strongly influence condensate stability. Many PPII helices have a low content of proline residues, which hinders their identification. Recently, we characterized the NMR spectral properties of a Gly‐rich, Pro‐poor protein composed of six PPII helices. Based on those results, we predicted that many Gly‐rich segments may form PPII helices and interact with PPII‐binding domains. This prediction is being tested and could join the palette of verified interactions contributing to biomolecular condensate formation. [ABSTRACT FROM AUTHOR]

The single‐stranded DNA binding protein (SSB) is essential to all aspects of DNA metabolism in bacteria. This protein performs two distinct, but closely intertwined and indispensable functions in the cell. SSB binds to single‐stranded DNA (ssDNA) and at least 20 partner proteins resulting in their regulation. These partners comprise a family of genome guardians known as the SSB interactome. Essential to interactome regulation is the linker/OB‐fold network of interactions. This network of interactions forms when one or more PXXP motifs in the linker of SSB bind to an OB‐fold in a partner, with interactome members involved in competitive binding between the linker and ssDNA to their OB‐fold. Consequently, when linker‐binding occurs to an OB‐fold in an interactome partner, proteins are loaded onto the DNA. When linker/OB‐fold interactions occur between SSB tetramers, cooperative ssDNA‐binding results, producing a multi‐tetrameric complex that rapidly protects the ssDNA. Within this SSB‐ssDNA complex, there is an extensive and dynamic network of linker/OB‐fold interactions that involves multiple tetramers bound contiguously along the ssDNA lattice. The dynamic behavior of these tetramers which includes binding mode changes, sliding as well as DNA wrapping/unwrapping events, are likely coupled to the formation and disruption of linker/OB‐fold interactions. This behavior is essential to facilitating downstream DNA processing events. As OB‐folds are critical to the essence of the linker/OB‐fold network of interactions, and they are found in multiple interactome partners, the SSB interactome is classified as the first family of prokaryotic, oligosaccharide/oligonucleotide binding fold (OB‐fold) genome guardians. [ABSTRACT FROM AUTHOR]

Src kinase belongs to the family of Src‐related nonreceptor tyrosine kinases. Because of its physiological role in cell growth and proliferation, its activity is strictly controlled by several mechanisms. Nevertheless, in viral Src kinase (v‐Src) some of these mechanisms fail, and its uncontrolled activity is responsible for the occurrence of cancer. Here, the crystal structures of three SH3‐domain mutants of v‐Src were determined to unveil the effects of these oncogenic mutations in this regulatory domain. Mutations in the n‐Src and distal loops have a low impact on the overall structure of the domain and its capacity to form intertwined dimers. However, mutations in the RT loop compromise the stability of the domain and make the protein very prone to aggregation. Additionally, these mutations prevent the formation of intertwined dimers. The results show a synergistic effect between mutations in the RT loop and those in the n‐Src and distal loops. Analysis of the structures of the v‐Src SH3‐domain mutants and the closed inactive conformation of cellular Src kinase (c‐Src) point to a loss of the interactions that are required to establish the compact inactive form of the kinase. Nevertheless, an analysis of structures of the c‐Src SH3 domain complexed with class I and II peptides points to minor changes in the interactions between the v‐Src SH3 domain and these peptides. In this way, the structures reported here indicate that mutations in the RT loop might impair the kinase regulation mechanism without affecting the recognition of short proline‐rich motifs in the target proteins of the kinase, thus explaining the oncogenic behaviour of the protein. [ABSTRACT FROM AUTHOR]

In addition to mediating nuclear transcription, PR mediates extranuclear functions mainly through the PR polyproline domain (PPD) interaction with the SH3 domain of cytoplasmic signaling molecules. PR-PPD-SH3 interaction inhibits EGF-mediated signaling and decreases lung cancer cell proliferation. Grb2 is an essential adaptor molecule with an SH2 domain flanked by two SH3 domains. In this study, we examined whether PR, through interaction between PR-PPD and Grb2-SH3, can interact with Grb2 in cells and breast cancer tissues. Our previous study shows that interaction between PR-PPD and Grb2 could interfere with cytoplasmic signaling and lead to inhibition of EGF-mediated signaling. GST-pulldown analysis shows that PR-PPD specifically interacts with the SH3 domains of Grb2. Immunofluorescence staining shows colocalization of PR and Grb2 in both the nucleus and cytoplasm in BT-474 breast cancer cells. Using Bimolecular Fluorescence Complementation (BiFC) analysis, we show that PR and Grb2 interact in breast cancer cells through the Grb2-SH3 domain. Proximity Ligation Assay (PLA) analysis of 43 breast cancer specimens shows that PR-Grb2 interaction is associated with low histological stage and negatively correlates with lymph node invasion and metastasis in breast cancer. These results, together with our previous findings, suggest that PR-PPD interaction with Grb2 plays an essential role in PR-mediated growth factor signaling inhibition and could contribute significantly to better prognosis in PR- and Grb2-positive breast cancer. Our finding provides a basis for additional studies to explore a novel therapeutic strategy for cancer treatment. • PR through its polyproline domain (PPD) has been shown to inhibit cell proliferation and EGF signaling. • Grb2, an important signaling molecule, contain an SH3 domain that can interacts with PR PPD. • PR through its PPD interacts with Grb2-SH3 domain in vitro and in cells. • PR-Grb2 interactions were detected in clinical breast cancer tissue samples. • Breast cancers that showed PR-Grb2 interaction are correlated with less aggressive tumors and better prognosis. [ABSTRACT FROM AUTHOR]

SAM68 is an mRNA-binding protein involved in mRNA processing in the nucleus that forms membraneless compartments called SAM68 Nuclear Bodies (SNBs). We found that intersectin 1 (ITSN1), a multidomain scaffold protein harboring five soluble SH3 domains, interacts with SAM68 proline-rich motifs (PRMs) surrounded by self-adhesive low complexity domains. While SAM68 is poorly soluble in vitro, the interaction of ITSN1 SH3 domains and mRNA with SAM68 enhances its solubility. In HeLa cells, the interaction between the first ITSN1 SH3 domain (SH3A) and P0, the N-terminal PRM of SAM68, induces the dissociation of SNBs. In addition, we reveal the ability of another SH3 domain (SH3D) of ITSN1 to bind to mRNAs. ITSN1 and mRNA may thus act in concert to promote SAM68 solubilization, consistent with the absence of mRNA in SNBs in cells. Together, these results support the notion of a specific chaperoning of PRM-rich SAM68 within nuclear ribonucleoprotein complexes by ITSN1 that may regulate the processing of a fraction of nuclear mRNAs, notably SAM68-controlled splicing events related to higher neuronal functions or cancer progression. This observation may also serve as a putative model of the interaction between other PRM-rich RBPs and signaling proteins harboring SH3 domains. [ABSTRACT FROM AUTHOR]

Adaptor proteins play a pivotal role in cellular signaling mediating a multitude of protein-protein interaction critical for cellular homeostasis. Dysregulation of these interactions has been linked to the onset of various cancer pathologies and exploited by viral pathogens during host cell takeover. CrkL is an adaptor protein composed of an N-terminal SH2 domain followed by two SH3 domains that mediate interactions with diverse partners through the recognition of specific binding motifs. In this study, we employed proteomic peptide-phage display (ProP-PD) to comprehensively explore the short linear motif (SLiM)-based interactions of CrkL. Furthermore, we scrutinized how the binding affinity for selected peptides was influenced in the context of the full-length CrkL versus the isolated N-SH3 domain. Importantly, our results provided insights into SLiM-binding sites within previously reported interactors, as well as revealing novel human and viral ligands, expanding our understanding of the interactions mediated by CrkL and highlighting the significance of SLiM-based interactions in mediating adaptor protein function, with implications for cancer and viral pathologies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Skeletal muscle development and regeneration are tightly regulated processes. How the intracellular organization of muscle fibers is achieved during these steps is unclear. Here, we focus on the cellular and physiological roles of amphiphysin 2 (BIN1), a membrane remodeling protein mutated in both congenital and adult centronuclear myopathies (CNM), that is ubiquitously expressed and has skeletal muscle-specific isoforms. We created and characterized constitutive muscle-specific and inducible Bin1 homozygous and heterozygous knockout mice targeting either ubiquitous or muscle-specific isoforms. Constitutive Bin1-deficient mice died at birth from lack of feeding due to a skeletal muscle defect. T-tubules and other organelles were misplaced and altered, supporting a general early role for BIN1 in intracellular organization, in addition to membrane remodeling. Although restricted deletion of Bin1 in unchallenged adult muscles had no impact, the forced switch from the muscle-specific isoforms to the ubiquitous isoforms through deletion of the in-frame muscle-specific exon delayed muscle regeneration. Thus, ubiquitous BIN1 function is necessary for muscle development and function, whereas its muscle-specific isoforms fine tune muscle regeneration in adulthood, supporting that BIN1 CNM with congenital onset are due to developmental defects, whereas later onset may be due to regeneration defects.

Src, the prototype of Src family kinases (SFKs), is a modular protein consisting of SH4 (SH4) and unique (UD) domains in an N-terminal intrinsically disordered region (IDR), and SH3, SH2, and kinase (KD) folded domains conserved among SFKs. Src functions as a pleiotropic signaling hub in proliferating and post-mitotic cells, and it is related to cancer and neurological diseases. However, its regulatory mechanism is unclear because the existing canonical model is derived from crystallographic analyses of folded constructs lacking the IDR. This work reviews nuclear magnetic resonance analyses of partially structured lipid-binding segments in the flexible UD and the fuzzy intramolecular complex (FIMC) comprising IDR and SH3 domains, which interacts with lipid membranes and proteins. Furthermore, recently determined IDR-related Src characteristics are discussed, including dimerization, SH4/KD intramolecular fastener bundling of folded domains, and the sorting of adhesive structures. Finally, the modulatory roles of IDR phosphorylation in Src activities involving the FIMC are explored. The new regulatory roles of IDRs are integrated with the canonical model to elucidate the functions of full-length Src. This review presents new aspects of Src regulation, and provides a future direction for studies on the structure and function of Src, and their implications for pathological processes.

Human neuron-specific PACSIN1 plays a key role in synaptic vesicle recycling and endocytosis, as well as reorganization of the microtubule dynamics to maintain axonal plasticity. PACSIN1 contains a highly conserved C-terminal SH3 domain and an F-bar domain at its N-terminus. Due to its remarkable interaction network, PACSIN1 plays a central role in key neuronal functions. Here, we present a robust backbone and side-chain assignment of PACSIN1 SH3 domain based on 2D [1H,15N] HSQC or HMQC, and 3D BEST-HNCO, -HNCACB, -HN(CO)CACB, -HN(CA)CO, and standard (H)CC(CO)NH, HN(CA)NNH, HN(COCA)NH, HBHANNH, HNHA, HBHA(CO)NH, H(CC)(CO)NH, HCCH-TOCSY, that covers 96% for all 13CO, 13Cα and 13Cβ, 28% of 13Cγδε, and 95% of 1HN and 15N chemical shifts. Modelling based on sequence homology with a known related structure, and chemical shift-based secondary structure predictions, identified the presence of five β-strands linked by flexible loops. Taken together, these results open up new avenues to investigate and develop new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Metal binding to sites engineered in proteins can provide an increase in their stability and facilitate new functions. Besides the sites introduced in purpose, sometimes they are present accidentally as a consequence of the expression system used to produce the protein. This happens with the copper- and nickel-binding (ATCUN) motif generated by the amino-terminal residues Gly-Ser-His. This ATCUN motif is fortuitously present in many proteins, but how it affects the structural and biophysical characterization of the proteins has not been studied. In this work, we have compared the structure and biophysical properties of a small modular domain, the SH3 domain of the c-Src tyrosine kinase, cloned with and without an ATCUN motif at the N terminus. At pH 7.0, the SH3 domain with the ATCUN motif binds nickel with a binding constant Ka = 28.0 ± 3.0 mM−1. The formation of the nickel complex increases the thermal and chemical stability of the SH3 domain. A comparison of the crystal structures of the SH3 domain with and without the ATCUN motif shows that the binding of nickel does not affect the overall structure of the SH3 domain. In all crystal structures analyzed, residues Gly-Ser-His in complex with Ni2+ show a square planar geometry. The CD visible spectrum of the nickel complex shows that this geometry is also present in the solution. Therefore, our results not only show that the ATCUN motif might influence the biophysical properties of the protein, but also points to an advantageous stabilization of the protein with potential biotechnological applications. [ABSTRACT FROM AUTHOR]

This study presents the crystal structure of the N‐terminal SH3 (SH3N) domain of growth factor receptor‐bound protein 2 (Grb2) at 2.5 Å resolution. Grb2 is a small (215‐amino‐acid) adaptor protein that is widely expressed and involved in signal transduction/cell communication. The crystal structure of full‐length Grb2 has previously been reported (PDB entry 1gri). The structure of the isolated SH3N domain is consistent with the full‐length structure. The structure of the isolated SH3N domain was solved at a higher resolution (2.5 Å compared with 3.1 Å for the previously deposited structure) and made it possible to resolve some of the loops that were missing in the full‐length structure. In addition, interactions between the carboxy‐terminal region of the SH3N domain and the Sos1‐binding sites were observed in the structure of the isolated domain. Analysis of these interactions provided new information about the ligand‐binding properties of the SH3N domain of Grb2. [ABSTRACT FROM AUTHOR]

The Escherichia coli single‐strand DNA binding protein (SSB) is essential to viability where it functions to regulate SSB interactome function. Here it binds to single‐stranded DNA and to target proteins that comprise the interactome. The region of SSB that links these two essential protein functions is the intrinsically disordered linker. Key to linker function is the presence of three, conserved PXXP motifs that mediate binding to oligosaccharide‐oligonucleotide binding folds (OB‐fold) present in SSB and its interactome partners. Not surprisingly, partner OB‐fold deletions eliminate SSB binding. Furthermore, single point mutations in either the PXXP motifs or, in the RecG OB‐fold, obliterate SSB binding. The data also demonstrate that, and in contrast to the view currently held in the field, the C‐terminal acidic tip of SSB is not required for interactome partner binding. Instead, we propose the tip has two roles. First, and consistent with the proposal of Dixon, to regulate the structure of the C‐terminal domain in a biologically active conformation that prevents linkers from binding to SSB OB‐folds until this interaction is required. Second, as a secondary binding domain. Finally, as OB‐folds are present in SSB and many of its partners, we present the SSB interactome as the first family of OB‐fold genome guardians identified in prokaryotes. [ABSTRACT FROM AUTHOR]

The Feline calicivirus (FCV) leader of the capsid (LC) protein is a viroporin required for an efficient viral replication. It is responsible for the induction of the cytopathic effect during infection and for the mitochondrial apoptosis when expressed in CRFK cells. The LC viroporin sequence contains two conserved regions: a polycisteine sequence implicated in the formation of disulfide bonds and a polyproline region (PVVPP) at the carboxyl terminus with an unknown function. Polyproline regions are implicated in the interaction with SH3 domain containing proteins. To determine if the LC protein interacts with cellular proteins, immunoprecipitation (IP) and mass spectrometry (MS) assays were performed. We found 135 LC-interacting cellular proteins; among them, the Fucosyltransferase 8 (FUT8), an enzyme that catalyzes the addition of fucose in alpha 1-6 linkage to the first GlcNAc residue and has an SH3 domain, characterized by having a beta barrel fold consisting on six β-strands arranged as two tightly packed anti-parallel β sheets. We analyzed the in silico interaction of both proteins and we will study the relevance of this interaction in infected cells. [ABSTRACT FROM AUTHOR]

The scaffold protein Tks4 is a member of the p47phox-related organizer superfamily. It plays a key role in cell motility by being essential for the formation of podosomes and invadopodia. In addition, Tks4 is involved in the epidermal growth factor (EGF) signaling pathway, in which EGF induces the translocation of Tks4 from the cytoplasm to the plasma membrane. The evolutionarily-related protein p47phox and Tks4 share many similarities in their N-terminal region: a phosphoinositide-binding PX domain is followed by two SH3 domains (so called “tandem SH3”) and a proline-rich region (PRR). In p47phox, the PRR is followed by a relatively short, disordered C-terminal tail region containing multiple phosphorylation sites. These play a key role in the regulation of the protein. In Tks4, the PRR is followed by a third and a fourth SH3 domain connected by a long (~420 residues) unstructured region. In p47phox, the tandem SH3 domain binds the PRR while the first SH3 domain interacts with the PX domain, thereby preventing its binding to the membrane. Based on the conserved structural features of p47phox and Tks4 and the fact that an intramolecular interaction between the third SH3 and the PX domains of Tks4 has already been reported, we hypothesized that Tks4 is similarly regulated by autoinhibition. In this study, we showed, via fluorescence-based titrations, MST, ITC, and SAXS measurements, that the tandem SH3 domain of Tks4 binds the PRR and that the PX domain interacts with the third SH3 domain. We also investigated a phosphomimicking Thr-to-Glu point mutation in the PRR as a possible regulator of intramolecular interactions. Phosphatidylinositol-3-phosphate (PtdIns(3)P) was identified as the main binding partner of the PX domain via lipid-binding assays. In truncated Tks4 fragments, the presence of the tandem SH3, together with the PRR, reduced PtdIns(3)P binding, while the presence of the third SH3 domain led to complete inhibition.

Abstract Background Chronic myelogenous leukemia (CML) is a pluripotent hematopoietic stem cell disorder caused by the fusion of the BCR and ABL1 genes. Quantitative RT-PCR (qRT-PCR) is a routinely performed screening technique to identify BCR-ABL1 fusion genes, but a limitation of this method is its inability to recognize novel fusions that have not been previously characterized. Next-generation sequencing (NGS) is an effective and sensitive detection method for the determination of novel BCR-ABL1 fusion genes as well as previously characterized ones. The oncoprotein tyrosine kinase BCR-ABL1 is a constitutively active kinase involved in the activation of a number of signaling pathways, and it has been the therapeutic target for tyrosine kinase inhibitors (TKIs) such as imatinib. Reports have presented opposing viewpoints about the effect of the disrupted Src homology 3 (SH3) domain on TKI efficacy. Findings We here report that using NGS we identified a novel BCR-ABL1 fusion gene with breakpoints in the BCR intron 14 and the ABL1 intron 2, leading to partial deletion of its SH3 domain. In the present case, the patient received targeted therapy with the TKI imatinib at 400 mg/day and no adverse reaction was reported. The patient eventually entered remission with decreased proliferation of karyocytes and granulocytes. We also identified mutations in genes, including TP53, FLT3, ASXL1, SETBP1, CEBPA and CBL, that seemed to have an influence on the outcome of TKI therapy targeting the BCR-ABL1 protein. Conclusions Together with previously reported results, it is clear that the genetic heterogeneity of CML patients significantly affects the presentation of the disease and its progression and therefore should inform the design of the therapeutic strategy.

The Bin-Amphiphysin-Rvs (BAR) domain of endophilin binds to the cell membrane and shapes it into a tubular shape for endocytosis. Endophilin has a Src-homology 3 (SH3) domain at their C-terminal. The SH3 domain interacts with the proline-rich motif (PRM) that is found in proteins such as neural Wiskott-Aldrich syndrome protein (N-WASP). Here, we re-examined the binding sites of the SH3 domain of endophilin in N-WASP by machine learning-based prediction and identified the previously unrecognized binding site. In addition to the well-recognized PRM at the central proline-rich region, we found a PRM in front of the N-terminal WASP homology 1 (WH1) domain of N-WASP (NtPRM) as a binding site of the endophilin SH3 domain. Furthermore, the diameter of the membrane tubules in the presence of NtPRM mutant was narrower and wider than that in the presence of N-WASP and in its absence, respectively. Importantly, the NtPRM of N-WASP was involved in the membrane localization of endophilin A2 in cells. Therefore, the NtPRM contributes to the binding of endophilin to N-WASP in membrane remodeling., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Signal transduction, the ability of cells to perceive information from the surroundings and alter behavior in response, is an essential property of life. Studies on tyrosine kinase action fundamentally changed our concept of cellular regulation. The induced assembly of subcellular hubs via the recognition of local protein or lipid modifications by modular protein interactions is now a central paradigm in signaling. Such molecular interactions are mediated by specific protein interaction domains. The first such domain identified was the SH2 domain, which was postulated to be a reader capable of finding and binding protein partners displaying phosphorylated tyrosine side chains. The SH3 domain was found to be involved in the formation of stable protein sub-complexes by constitutively attaching to proline-rich surfaces on its binding partners. The SH2 and SH3 domains have thus served as the prototypes for a diverse collection of interaction domains that recognize not only proteins but also lipids, nucleic acids, and small molecules. It has also been found that particular SH2 and SH3 domains themselves might also bind to and rely on lipids to modulate complex assembly. Some lipid-binding properties of SH2 and SH3 domains are reviewed here.