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6 recordsLinked to original sources
PubMed2025

Structure-informed theoretical modeling defines principles governing avidity in bivalent protein interactions.

In signaling cascades, where domain-motif interactions tend to interact with relatively low affinity (allowing for reversibility), signaling proteins often encode multiple domains or motifs, which present the possibility of avidity - drastically increasing the interaction strength and duration as a result of multivalent binding. However, given the large combinatorial space, predicting and validating multivalent interactions that interact with avidity is a challenge. Here, we integrate mechanistic modeling, structure-based analysis, and experimental approaches as a framework for defining the conditions under which avidity plays a role. We explore the tandem SH2 domain family of interactions with bisphosphorylated partners as a multivalent archetype, which encompasses key secondary messengers in tyrosine kinase signaling networks. While certain multivalent interactions have been shown to be necessary in immune receptor recruitment of partners, bivalent recruitment of tandem SH2 domains more broadly is poorly understood. Theoretical modeling suggests that maximum avidity occurs with closely spaced or flexibly linked phosphotyrosine sites, combined with moderate monovalent affinities - exactly around the innate range of SH2 domain affinity. Surprisingly, despite sequence diversity, structure-based analysis showed remarkably conserved three-dimensional spacing between SH2 domains across all tandem SH2 families, which we corroborate experimentally, suggesting evolutionary optimization for avidity interactions. The combination of structure-based analysis of domain spacing with available monovalent experimental data appears to be sufficiently accurate to predict and rank order high affinity interactions of tandem SH2 domain recruitment to the EGFR C-terminal tail. These approaches lay the groundwork for larger utility in multivalent prediction and testing to help better understand protein interactions that drive cell signaling.

BLI↗
PubMed1979

An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates.

Polyoma T antigen immunoprecipitates contain a protein kinase-like activity which preferentially phosphorylates material of 50-60,000 daltons molecular weight. Phosphorylation is not diminished in extracts of polyoma tsA mutant-infected cells shifted to the nonpermissive temperature late in infection, conditions which inactivate the large T antigen. Phosphorylation is reduced or absent in cells infected with polyoma host range nontransforming (hr-t) mutants, which have defective small and medium T antigens. The major acceptor of phosphate is not the heavy chain of immunoglobulin, but appears to be the polyoma medium T antigen. The large T antigen is also phosphorylated, but usually to a lower specific activity. In terms of acid and alkali sensitivity and electrophoretic and chromatographic mobility in one and two dimensions, the phosphorylated residue behaves identically to phosphotyrosine and differently than phosphorylated serine, threonine, lysine and histidine.

Animals↗
PubMed1978

O4-(5'-uridylyl)tyrosine is the bond between the genome-linked protein and the RNA of poliovirus.

Virion RNA of poliovirus type 1 has been analyzed for the linkage between genome-protein VPg and the polyribonucleotide chain. Hydrolysis of the linkage with acid or alkali and enzymatic degradation lead to the conclusion that the bond is neither a phosphodiester such as nucleotidyl-(P-O)-serine (or threonine) nor a phosphoramidate such as nucleotidyl-(P-N)-amino acid. VPg-RNA can be iodinated by the Bolton and Hunter reagent [iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester] but not by the chloramine-T or lactoperoxidase procedures, an observation suggesting that VPg does not contain accessible tyrosine. However, VPg can be labeled with [3H]tyrosine in vivo. Hydrolysis of VPg-[32P]pUp with 5.6 M HCl at 110 degrees yielded 32P-labeled O4-(3'-phospho-5'-uridylyl)tyrosine that could be cleaved with micrococcal nuclease to O4-[32P]phosphotyrosine and uridine 3'-[32P]phosphate. These data establish that VPg is linked to the poliovirus genome by a bond between the O4 of tyrosine and the 5'-P atom of the terminal uridylic acid residue. The 5' end of polio genome RNA can now be described as VPg(Tyr-O)-pU-U-A-A-A-A-C-A-G.

Poliovirus↗
PubMed2025

CoDIAC: A comprehensive approach for interaction analysis reveals novel insights into SH2 domain function and regulation.

Protein domains are conserved structural and functional units that serve as building blocks of proteins. Through evolutionary expansion, domain families are represented by multiple members in diverse configurations with other domains, evolving new specificities for their interacting partners. Here, we develop a structure-based interface analysis to comprehensively map domain interfaces from experimental and predicted structures, including interfaces with macromolecules and intraprotein interfaces. We hypothesized that comprehensive contact mapping of domains could yield new insights into domain selectivity, conservation of domain-domain interfaces across proteins, and identify conserved post-translational modifications (PTMs), relative to interaction interfaces, allowing for the inference of specific effects due to PTMs or mutations. We applied this approach to the human SH2 domain family, a modular unit central to phosphotyrosine-mediated signaling, identifying a novel approach to understanding binding selectivity and evidence of coordinated regulation of SH2 domain binding interfaces by tyrosine and serine/threonine phosphorylation and acetylation. These findings suggest multiple signaling systems can regulate protein activity and SH2 domain interactions in a coordinated manner. We provide the extensive features of the human SH2 domain family and this modular approach as an open source Python package for COmprehensive Domain Interface Analysis of Contacts (CoDIAC).

SH2 domains↗
PubMed2025

SH2-mediated steric occlusion of the C2 domain regulates autoinhibition of SHIP1 inositol 5-phosphatase.

The Src homology 2 (SH2) domain containing inositol polyphosphate 5-phosphatase 1 (SHIP1) is an immune cell specific enzyme that regulates phosphatidylinositol-(3,4,5)-trisphosphate signaling at the plasma membrane following receptor activation. SHIP1 plays an important role in processes such as directed cell migration, endocytosis, and cortical membrane oscillations. Alterations in SHIP1 expression have been shown to perturb myeloid cell chemotaxis and differentiation. In the brain, SHIP1 regulate microglial cell behaviors, which has been linked to Alzheimer's disease. Understanding the structural and functional relationships of SHIP1 is critical for developing ways to modulate SHIP1 membrane localization and lipid phosphatase activity during immune cell signaling. Recently, we discovered that the N-terminal SH2 domain of SHIP1 suppresses lipid phosphatase activity. SHIP1 autoinhibition can be relieved through interactions with receptor-derived phosphotyrosine (pY) peptides presented on membranes or in solution. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) we identified intramolecular contacts between the N-terminal SH2 domain and CBL1 motif of the C2 domain that limit SHIP1 membrane localization and activity. Single molecule measurements of purified SHIP1 on supported lipid bilayers and in neutrophil-like cells support a model in which the SH2 domain blocks membrane binding of the central catalytic module. Mutations that disrupt autoinhibition enhance the membrane binding frequency and increase the catalytic efficiency of SHIP1. Although dimerization of SHIP1 enhances membrane localization and the apparent phosphatase activity, it is not required for SHIP1 autoinhibition. Overall, our results provide new insight concerning SHIP1's structural organization, membrane binding dynamics, and the mechanism of autoinhibition.

Journal Article↗
PubMed2026

Structure-informed theoretical modeling defines principles governing avidity in bivalent protein interactions.

In signaling cascades, signaling proteins often encode multiple domains or motifs, which presents the possibility for avidity -- where multivalent binding drastically increases interaction strength and duration. However, predicting and validating multivalent interactions that interact with avidity is a challenge. Here, we integrate mechanistic modeling, structure-based analysis, and experimental approaches as a framework for defining the conditions under which avidity plays a role. We explore the tandem SH2 domain family of interactions with bisphosphorylated partners as a multivalent archetype, which encompasses key secondary messengers in tyrosine kinase signaling networks. Theoretical modeling suggests that maximum avidity occurs with closely spaced tyrosine phosphorylation sites combined with moderate monovalent affinities - exactly around the innate range of SH2 domain affinity - or with phosphorylation sites separated by sufficiently flexible linkers. Surprisingly, despite sequence diversity, structure-based analysis showed relatively conserved three-dimensional spacing between SH2 domains across all tandem SH2 families, which we corroborate experimentally, suggesting evolutionary optimization for avidity interactions. The combination of structure-based analysis of domain spacing with available monovalent experimental data appears, along with iterative experimental refinement of biophysical parameters, can identify high affinity interactions of tandem SH2 domain recruitment to the EGFR C-terminal tail. Using these principles, we extended bivalent predictions into the full phosphoproteome space and structural parameterization of other partners of SH2 domain binding, providing resources and methods for more rapid expansion of bivalent analysis. These approaches lay the groundwork for larger utility in multivalent prediction and testing to help better understand protein interactions that drive cell signaling.

BLI↗