MVsim is a toolset for quantifying and designing multivalent interactions.

Arising through multiple binding elements, multivalency can specify the avidity, duration, cooperativity, and selectivity of biomolecular interactions, but quantitative prediction and design of these properties has remained challenging. Here we present MVsim, an application suite built around a configurational network model of multivalency to facilitate the quantification, design, and mechanistic evaluation of multivalent binding phenomena through a simple graphical user interface. To demonstrate the utility and versatility of MVsim, we first show that both monospecific and multispecific multivalent ligand-receptor interactions, with their noncanonical binding kinetics, can be accurately simulated. Further, to illustrate the conceptual insights into multivalent systems that MVsim can provide, we apply it to quantitatively predict the ultrasensitivity and performance of multivalent-encoded protein logic gates, evaluate the inherent programmability of multispecificity for selective receptor targeting, and extract rate constants of conformational switching for the SARS-CoV-2 spike protein and model its binding to ACE2 as well as multivalent inhibitors of this interaction. MVsim and instructional tutorials are freely available at https://sarkarlab.github.io/MVsim/ .

MVsim : a toolset for quantifying and designing multivalent interactions.

Arising through multiple binding elements, multivalency can specify the avidity, duration, cooperativity, and selectivity of biomolecular interactions, but quantitative prediction and design of these properties has remained challenging. Here we present MVsim , an application suite built around a configurational network model of multivalency to facilitate the quantification, design, and mechanistic evaluation of multivalent binding phenomena through a simple graphical user interface. To demonstrate the utility and versatility of MVsim , we first show that both monospecific and multispecific multivalent ligand-receptor interactions, with their noncanonical binding kinetics, can be accurately simulated. We then quantitatively predict the ultrasensitivity and performance of multivalent-encoded protein logic gates, evaluate the inherent programmability of multispecificity for selective receptor targeting, and extract rate constants of conformational switching for the SARS-CoV-2 spike protein and model its binding to ACE2 as well as multivalent inhibitors of this interaction. MVsim is freely available at https://sarkarlab.github.io/MVsim/ .

Mechanisms of noncanonical binding dynamics in multivalent protein-protein interactions.

Protein multivalency can provide increased affinity and specificity relative to monovalent counterparts, but these emergent biochemical properties and their mechanistic underpinnings are difficult to predict as a function of the biophysical properties of the multivalent binding partners. Here, we present a mathematical model that accurately simulates binding kinetics and equilibria of multivalent protein-protein interactions as a function of the kinetics of monomer-monomer binding, the structure and topology of the multidomain interacting partners, and the valency of each partner. These properties are all experimentally or computationally estimated a priori, including approximating topology with a worm-like chain model applicable to a variety of structurally disparate systems, thus making the model predictive without parameter fitting. We conceptualize multivalent binding as a protein-protein interaction network: ligand and receptor valencies determine the number of interacting species in the network, with monomer kinetics and structural properties dictating the dynamics of each species. As predicted by the model and validated by surface plasmon resonance experiments, multivalent interactions can generate several noncanonical macroscopic binding dynamics, including a transient burst of high-energy configurations during association, biphasic equilibria resulting from interligand competition at high concentrations, and multiexponential dissociation arising from differential lifetimes of distinct network species. The transient burst was only uncovered when extending our analysis to trivalent interactions due to the significantly larger network, and we were able to predictably tune burst magnitude by altering linker rigidity. This study elucidates mechanisms of multivalent binding and establishes a framework for model-guided analysis and engineering of such interactions.

Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.

Membrane-less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher-order assemblies influences the recruitment of the speckle-type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin-3-RING ubiquitin ligase (CRL3) substrate adaptor, self-associates into higher-order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild-type SPOP localizes to liquid nuclear speckles, self-association-deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB-mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration-dependent populations of the resulting oligomeric species. Higher-order oligomerization of SPOP stimulates CRL3(SPOP) ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher-order protein self-association may be a general mechanism to concentrate functional components in membrane-less cellular bodies.

Prostate cancer. Ubiquitylome analysis identifies dysregulation of effector substrates in SPOP-mutant prostate cancer.

Cancer genome characterization has revealed driver mutations in genes that govern ubiquitylation; however, the mechanisms by which these alterations promote tumorigenesis remain incompletely characterized. Here, we analyzed changes in the ubiquitin landscape induced by prostate cancer-associated mutations of SPOP, an E3 ubiquitin ligase substrate-binding protein. SPOP mutants impaired ubiquitylation of a subset of proteins in a dominant-negative fashion. Of these, DEK and TRIM24 emerged as effector substrates consistently up-regulated by SPOP mutants. We highlight DEK as a SPOP substrate that exhibited decreases in ubiquitylation and proteasomal degradation resulting from heteromeric complexes of wild-type and mutant SPOP protein. DEK stabilization promoted prostate epithelial cell invasion, which implicated DEK as an oncogenic effector. More generally, these results provide a framework to decipher tumorigenic mechanisms linked to dysregulated ubiquitylation.

Adaptor protein self-assembly drives the control of a cullin-RING ubiquitin ligase.

The E3 ligases recruit substrate proteins for targeted ubiquitylation. Recent insights into the mechanisms of ubiquitylation demonstrate that E3 ligases can possess active regulatory properties beyond those of a simple assembly scaffold. Here, we describe the dimeric structure of the E3 ligase adaptor protein SPOP (speckle-type POZ protein) in complex with the N-terminal domain of Cul3 at 2.4 Å resolution. We find that SPOP forms large oligomers that can form heteromeric species with the closely related paralog SPOPL. In combination, SPOP and SPOPL (SPOP-like) form a molecular rheostat that can fine-tune E3 ubiquitin ligase activity by affecting the oligomeric state of the E3 complex. We propose that adaptor protein self-assembly provides a graded level of regulation of the SPOP/Cul3 E3 ligase toward its multiple protein substrates.

Kelch-like homologue 9 mutation is associated with an early onset autosomal dominant distal myopathy.

Distal myopathies are a heterogeneous group of disorders characterized by progressive weakness and muscular atrophy, beginning in distal limb muscles and affecting proximal limb muscles at a later stage. We studied a large German kindred with 10 affected members. Weakness and atrophy of the anterior tibial muscles started between the ages of 8 and 16 years, followed by atrophy of intrinsic hand muscles. Progression was slow, and patients retained the ability to walk until the seventh decade. Serum creatinine kinase levels were increased in the range of 150-1400 U/l. Muscle biopsies showed myopathic changes, whereas immunohistochemistry showed normal expression of marker proteins for muscular dystrophies. Patients had reduced sensation with stocking-glove distribution in the distal limbs in later life. Nerve conduction studies revealed no evidence of neuropathy. Genome-wide linkage analysis in this family revealed a new locus for distal myopathy at 9p21.2-p22.3 (multipoint logarithm of the odds ratio=4.21). By positional cloning we found a heterozygous mutation L95F in the Kelch-like homologue 9 gene, encoding a bric-a-brac Kelch protein. Molecular modelling indicated that the mutation may interfere with the interaction of the bric-a-brac domain with Cullin 3. Coimmunoprecipitation experiments confirmed that the mutation reduces association with Cullin 3 in the Kelch-like homologue 9-Cullin 3-E3 ubiquitin ligase complex, which is involved in ubiquitin-dependent protein degradation. We identified a unique form of early onset autosomal dominant distal myopathy which is associated with a Kelch-like homologue 9 mutation and interferes with normal skeletal muscle through a novel pathogenetic mechanism.

Beads-on-a-string, characterization of ETS-1 sumoylated within its flexible N-terminal sequence.

Sumoylation regulates the activities of several members of the ETS transcription factor family. To provide a molecular framework for understanding this regulation, we have characterized the conjugation of Ets-1 with SUMO-1. Ets-1 is modified in vivo predominantly at a consensus sumoylation motif containing Lys-15. This lysine is located within the unstructured N-terminal segment of Ets-1 preceding its PNT domain. Using NMR spectroscopy, we demonstrate that the Ets-1 sumoylation motif associates with the substrate binding site on the SUMO-conjugating enzyme UBC9 (K(d) approximately 400 microm) and that the PNT domain is not involved in this interaction. Ets-1 with Lys-15 mutated to an arginine still binds UBC9 with an affinity similar to the wild type protein, but is no longer sumoylated. NMR chemical shift and relaxation measurements reveal that the covalent attachment of mature SUMO-1, via its flexible C-terminal Gly-97, to Lys-15 of Ets-1 does not perturb the structure or dynamic properties of either protein. Therefore sumoylated Ets-1 behaves as "beads-on-a-string" with the two proteins tethered by flexible polypeptide segments containing the isopeptide linkage. Accordingly, SUMO-1 may mediate interactions of Ets-1 with signaling or transcriptional regulatory macromolecules by acting as a structurally independent docking module, rather than through the induction of a conformational change in either protein upon their covalent linkage. We also hypothesize that the flexibility of the linking polypeptide sequence may be a general feature contributing to the recognition of SUMO-modified proteins by their downstream effectors.

Structural and dynamic independence of isopeptide-linked RanGAP1 and SUMO-1.

Although sumoylation regulates a diverse and growing number of recognized biological processes, the molecular mechanisms by which the covalent attachment of the ubiquitin-like protein SUMO can alter the properties of a target protein remain to be established. To address this question, we have used NMR spectroscopy to characterize the complex of mature SUMO-1 with the C-terminal domain of human RanGAP1. Based on amide chemical shift and 15N relaxation measurements, we show that the C terminus of SUMO-1 and the loop containing the consensus sumoylation site in RanGAP1 are both conformationally flexible. Furthermore, the overall structure and backbone dynamics of each protein remain unchanged upon the covalent linkage of Lys524 in RanGAP1 to the C-terminal Gly97 of SUMO-1. Therefore, SUMO-1 and RanGAP1 behave as "beads-on-a-string," connected by a flexible isopeptide tether. Accordingly, the sumoylation-dependent interaction of RanGAP1 with the nucleoporin RanBP2 may arise through the bipartite recognition of both RanGAP1 and SUMO-1 rather than through a new binding surface induced in either individual protein upon their covalent linkage. We hypothesize that this conformational flexibility may be a general feature contributing to the recognition of ubiquitin-like modified proteins by their downstream effector machineries.