Matteo Lambrughi

PyInteraph: a framework for the analysis of interaction networks in structural ensembles of proteins.

In the last years, a growing interest has been gathering around the ability of Molecular Dynamics (MD) to provide insight into the paths of long-range structural communication in biomolecules. The knowledge of the mechanisms related to structural communication helps in the rationalization in atomistic details of the effects induced by mutations, ligand binding, and the intrinsic dynamics of proteins. We here present PyInteraph, a tool for the analysis of structural ensembles inspired by graph theory. PyInteraph is a software suite designed to analyze MD and structural ensembles with attention to binary interactions between residues, such as hydrogen bonds, salt bridges, and hydrophobic interactions. PyInteraph also allows the different classes of intra- and intermolecular interactions to be represented, combined or alone, in the form of interaction graphs, along with performing network analysis on the resulting interaction graphs. The program also integrates the network description with a knowledge-based force field to estimate the interaction energies between side chains in the protein. It can be used alone or together with the recently developed xPyder PyMOL plugin through an xPyder-compatible format. The software capabilities and associated protocols are here illustrated by biologically relevant cases of study. The program is available free of charge as Open Source software via the GPL v3 license at http://linux.btbs.unimib.it/pyinteraph/.

Communication Routes in ARID Domains between Distal Residues in Helix 5 and the DNA-Binding Loops

ARID is a DNA-binding domain involved in several transcriptional regulatory processes, including cell-cycle regulation and embryonic development. ARID domains are also targets of the Human Cancer Protein Interaction Network. Little is known about the molecular mechanisms related to conformational changes in the family of ARID domains. Thus, we have examined their structural dynamics to enrich the knowledge on this important family of regulatory proteins. In particular, we used an approach that integrates atomistic simulations and methods inspired by graph theory. To relate these properties to protein function we studied both the free and DNA-bound forms. The interaction with DNA not only stabilizes the conformations of the DNA-binding loops, but also strengthens pre-existing paths in the native ARID ensemble for long-range communication to those loops. Residues in helix 5 are identified as critical mediators for intramolecular communication to the DNA-binding regions. In particular, we identified a distal tyrosine that plays a key role in long-range communication to the DNA-binding loops and that is experimentally known to impair DNA-binding. Mutations at this tyrosine and in other residues of helix 5 are also demonstrated, by our approach, to affect the paths of communication to the DNA-binding loops and alter their native dynamics. Overall, our results are in agreement with a scenario in which ARID domains exist as an ensemble of substates, which are shifted by external perturbation, such as the interaction with DNA. Conformational changes at the DNA-binding loops are transmitted long-range by intramolecular paths, which have their heart in helix 5.

Intramolecular interactions stabilizing compact conformations of the intrinsically disordered kinase-inhibitor domain of Sic1: a molecular dynamics investigation

Cyclin-dependent kinase inhibitors (CKIs) are key regulatory proteins of the eukaryotic cell cycle, which modulate cyclin-dependent kinase (Cdk) activity. CKIs perform their inhibitory effect by the formation of ternary complexes with a target kinase and its cognate cyclin. These regulators generally belong to the class of intrinsically disordered proteins (IDPs), which lack a well-defined and organized three-dimensional (3D) structure in their free state, undergoing folding upon binding to specific partners. Unbound IDPs are not merely random-coil structures, but can present intrinsically folded structural units (IFSUs) and collapsed conformations. These structural features can be relevant to protein function in vivo. The yeast CKI Sic1 is a 284-amino acid IDP that binds to Cdk1 in complex with the Clb5,6 cyclins, preventing phosphorylation of G1 substrates and, therefore, entrance to the S phase. Sic1 degradation, triggered by multiple phosphorylation events, promotes cell-cycle progression. Previous experimental studies pointed out a propensity of Sic1 and its isolated domains to populate both extended and compact conformations. The present contribution provides models for compact conformations of the Sic1 kinase-inhibitory domain (KID) by all-atom molecular dynamics (MD) simulations in explicit solvent and in the absence of interactors. The results are integrated by spectroscopic and spectrometric data. Helical IFSUs are identified, along with networks of intramolecular interactions. The results identify a group of putative hub residues and networks of electrostatic interactions, which are likely to be involved in the stabilization of the globular states.

E2 superfamily of ubiquitin-conjugating enzymes: constitutively active or activated through phosphorylation in the catalytic cleft.

Protein phosphorylation is a modification that offers a dynamic and reversible mechanism to regulate the majority of cellular processes. Numerous diseases are associated with aberrant regulation of phosphorylation-induced switches. Phosphorylation is emerging as a mechanism to modulate ubiquitination by regulating key enzymes in this pathway. The molecular mechanisms underpinning how phosphorylation regulates ubiquitinating enzymes, however, are elusive. Here, we show the high conservation of a functional site in E2 ubiquitin-conjugating enzymes. In catalytically active E2s, this site contains aspartate or a phosphorylatable serine and we refer to it as the conserved E2 serine/aspartate (CES/D) site. Molecular simulations of substrate-bound and -unbound forms of wild type, mutant and phosphorylated E2s, provide atomistic insight into the role of the CES/D residue for optimal E2 activity. Both the size and charge of the side group at the site play a central role in aligning the substrate lysine toward E2 catalytic cysteine to control ubiquitination efficiency. The CES/D site contributes to the fingerprint of the E2 superfamily. We propose that E2 enzymes can be divided into constitutively active or regulated families. E2s characterized by an aspartate at the CES/D site signify constitutively active E2s, whereas those containing a serine can be regulated by phosphorylation.

A comparative study of Whi5 and retinoblastoma proteins: from sequence and structure analysis to intracellular networks

Cell growth and proliferation require a complex series of tight-regulated and well-orchestrated events. Accordingly, proteins governing such events are evolutionary conserved, even among distant organisms. By contrast, it is more singular the case of “core functions” exerted by functional analogous proteins that are not homologous and do not share any kind of structural similarity. This is the case of proteins regulating the G1/S transition in higher eukaryotes–i.e., the retinoblastoma (Rb) tumor suppressor Rb—and budding yeast, i.e., Whi5. The interaction landscape of Rb and Whi5 is quite large, with more than one hundred proteins interacting either genetically or physically with each protein. The Whi5 interactome has been used to construct a concept map of Whi5 function and regulation. Comparison of physical and genetic interactors of Rb and Whi5 allows highlighting a significant core of conserved, common functionalities associated with the interactors indicating that structure and function of the network—rather than individual proteins—are conserved during evolution. A combined bioinformatics and biochemical approach has shown that the whole Whi5 protein is highly disordered, except for a small region containing the protein family signature. The comparison with Whi5 homologs from Saccharomycetales has prompted the hypothesis of a modular organization of structural disorder, with most evolutionary conserved regions alternating with highly variable ones. The finding of a consensus sequence points to the conservation of a specific phosphorylation rhythm along with two disordered sequence motifs, probably acting as phosphorylation-dependent seeds in Whi5 folding/unfolding. Thus, the widely disordered Whi5 appears to act as a hierarchical, “date hub” that has evolutionary assayed an original way of modular organization before being supplanted by the globular, multi-domain structured Rb, more suitable to cover the role of a “party hub”.

The conformational ensemble of the disordered and aggregation-protective 182–291 region of ataxin-3

BACKGROUND Intrinsically disordered proteins (IDPs) are an emerging part of structural biology that has challenged the classic paradigm of structure-function relationship. Indeed, IDPs have been associated with different physiological functions and associated with several pathologies, such as polyglutamine (polyQ) related diseases. Ataxin-3 (AT3) is the smallest polyQ protein, composed by the N-terminal folded Josephin domain (JD), which is amyloidogenic on its own, and a C-terminal unstructured part. The disordered region between the polyQ and the JD, AT3182-291 plays a key role in the development of the disease. METHODS We integrated different biophysical experimental techniques, atomistic explicit-solvent molecular dynamics (MD) simulations and graph theory to study AT3182-291 structure. RESULTS AT3182-291 is a monomeric intrinsically disordered (ID) domain in solution and it is characterized by different conformational states, ascribable to pre-molten globule populations with different degrees of compactness. If isolated, it decreases the aggregation of the entire AT3. CONCLUSIONS We provided the first structural description of an ID domain associated to a polyQ protein and we also showed that it exerts protective effects against AT3 aggregation. This effect is likely to be induced by intermolecular interactions between AT3 and the ubiquitin-interacting motifs of AT3182-291. Electrostatic interactions play a pivotal role in regulating the topology and tertiary propensity of the fragment and hub residues have been identified. GENERAL SIGNIFICANCE Synergistic use of atomistic simulations and biophysical techniques should be more generally applied to the study of IDPs. Since ID domains and polyQ-proteins are intimately connected, the study here provided can be of interest for other members of the group.

Catalytic Mechanism of Fungal Lytic Polysaccharide Monooxygenases Investigated by First-Principles Calculations

Lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes that facilitate the degradation of recalcitrant polysaccharides by the oxidative cleavage of glycosidic bonds. They are gaining rapidly increasing attention as key players in biomass conversion, especially for the production of second-generation biofuels. Elucidation of the detailed mechanism of the LPMO reaction is a major step toward the assessment and optimization of LPMO efficacy in industrial biotechnology, paving the way to utilization of sustainable fuel sources. Here, we used density functional theory calculations to study the reaction pathways suggested to date, exploiting a very large active-site model for a fungal AA9 LPMO and using a celloheptaose unit as a substrate mimic. We identify a copper oxyl intermediate as being responsible for H-atom abstraction from the substrate, followed by a rapid, water-assisted hydroxyl rebound, leading to substrate hydroxylation.

The Mutational Landscape of the Oncogenic MZF1 SCAN Domain in Cancer

SCAN domains in zinc-finger transcription factors are crucial mediators of protein-protein interactions. Up to 240 SCAN-domain encoding genes have been identified throughout the human genome. These include cancer-related genes, such as the myeloid zinc finger 1 (MZF1), an oncogenic transcription factor involved in the progression of many solid cancers. The mechanisms by which SCAN homo- and heterodimers assemble and how they alter the transcriptional activity of zinc-finger transcription factors in cancer and other diseases remain to be investigated. Here, we provide the first description of the conformational ensemble of the MZF1 SCAN domain cross-validated against NMR experimental data, which are probes of structure and dynamics on different timescales. We investigated the protein-protein interaction network of MZF1 and how it is perturbed in different cancer types by the analyses of high-throughput proteomics and RNASeq data. Collectively, we integrated many computational approaches, ranging from simple empirical energy functions to all-atom microsecond molecular dynamics simulations and network analyses to unravel the effects of cancer-related substitutions in relation to MZF1 structure and interactions.

An optimal distance cutoff for contact-based Protein Structure Networks using side-chain centers of mass

Proteins are highly dynamic entities attaining a myriad of different conformations. Protein side chains change their states during dynamics, causing clashes that are propagated at distal sites. A convenient formalism to analyze protein dynamics is based on network theory using Protein Structure Networks (PSNs). Despite their broad applicability, few efforts have been devoted to benchmarking PSN methods and to provide the community with best practices. In many applications, it is convenient to use the centers of mass of the side chains as nodes. It becomes thus critical to evaluate the minimal distance cutoff between the centers of mass which will provide stable network properties. Moreover, when the PSN is derived from a structural ensemble collected by molecular dynamics (MD), the impact of the MD force field has to be evaluated. We selected a dataset of proteins with different fold and size and assessed the two fundamental properties of the PSN, i.e. hubs and connected components. We identified an optimal cutoff of 5 Å that is robust to changes in the force field and the proteins. Our study builds solid foundations for the harmonization and standardization of the PSN approach.

Hybrid Voronoi diagrams, their computation and reduction for applications in computational biochemistry

Geometric models of molecular structures are often described as a set of balls, where balls represent individual atoms. The ability to describe and explore the empty space among these balls is important, e.g., in the analysis of the interaction of enzymes with substrates, ligands and solvent molecules. Voronoi diagrams from the field of computational geometry are often used here, because they provide a mathematical description of how the whole space can be divided into regions assigned to individual atoms. This paper introduces a combination of two different types of Voronoi diagrams into a new hybrid Voronoi diagram - one part of this diagram belongs to the additively weighted (aw-Voronoi) diagram and the other to the power diagram. The boundary between them is controlled by a user-defined constant (the probe radius). Both parts are computed by different algorithms, which are already known. The reduced aw-Voronoi diagram is then obtained by removing the power diagram part from the hybrid diagram. Reduced aw-Voronoi diagrams are perfectly tailored for the analysis of dynamic molecular structures, their computation is faster and storage requirements are lower than in the case of complete aw-Voronoi diagrams. Here, we showed their application to key proteins in cancer research such as p53 and ARID proteins as case study. We identified a biologically relevant cavity in p53 structural ensembles generated by molecular dynamics simulations and analyzed its accessibility, attesting the potential of our approach. This method is relevant for cancer research since it permits to depict a dynamical view of cavities and pockets in proteins that could be affected by mutations in the disease. Our approach opens novel prospects for the study of cancer-related proteins by molecular simulations and the identification of novel targets for drug design.