Dissecting Structure-Encoded Determinants of Allosteric Cross-Talk between Post-Translational Modification Sites in the Hsp90 Chaperones

Post-translational modifications (PTMs) represent an important regulatory instrument that modulates structure, dynamics and function of proteins. The large number of PTM sites in the Hsp90 proteins that are scattered throughout different domains indicated that synchronization of multiple PTMs through a combinatorial code can be invoked as an important mechanism to orchestrate diverse chaperone functions and recognize multiple client proteins. In this study, we have combined structural and coevolutionary analysis with molecular simulations and perturbation response scanning analysis of the Hsp90 structures to characterize functional role of PTM sites in allosteric regulation. The results reveal a small group of conserved PTMs that act as global mediators of collective dynamics and allosteric communications in the Hsp90 structures, while the majority of flexible PTM sites serve as sensors and carriers of the allosteric structural changes. This study provides a comprehensive structural, dynamic and network analysis of PTM sites across Hsp90 proteins, identifying specific role of regulatory PTM hotspots in the allosteric mechanism of the Hsp90 cycle. We argue that plasticity of a combinatorial PTM code in the Hsp90 may be enacted through allosteric coupling between effector and sensor PTM residues, which would allow for timely response to structural requirements of multiple modified enzymes

Scop3P : a comprehensive resource of human phosphosites within their full context

Protein phosphorylation is a key post-translational modification in many biological processes and is associated to human diseases such as cancer and metabolic disorders. The accurate identification, annotation, and functional analysis of phosphosites are therefore crucial to understand their various roles. Phosphosites are mainly analyzed through phosphoproteomics, which has led to increasing amounts of publicly available phosphoproteomics data. Several resources have been built around the resulting phosphosite information, but these are usually restricted to the protein sequence and basic site metadata. What is often missing from these resources, however, is context, including protein structure mapping, experimental provenance information, and biophysical predictions. We therefore developed Scop3P: a comprehensive database of human phosphosites within their full context. Scop3P integrates sequences (UniProtKB/Swiss-Prot), structures (PDB), and uniformly reprocessed phosphoproteomics data (PRIDE) to annotate all known human phosphosites. Furthermore, these sites are put into biophysical context by annotating each phosphoprotein with per-residue structural propensity, solvent accessibility, disordered probability, and early folding information. Scop3P, available at https://iomics.ugent.be/scop3p, presents a unique resource for visualization and analysis of phosphosites and for understanding of phosphosite structure–function relationships

The evolution, modifications and interactions of proteins and RNAs

Proteins and RNAs are two of the most versatile macromolecules that carry out almost all functions within living organisms. In this thesis I have explored evolutionary and regulatory aspects of proteins and RNAs by studying their structures, modifications and interactions. In the first chapter of my thesis I investigate domain atrophy, a term I coined to describe large-scale deletions of core structural elements within protein domains. By looking into truncated domain boundaries across several domain families using Pfam, I was able to identify rare cases of domains that showed atrophy. Given that even point mutations can be deleterious, it is surprising that proteins can tolerate such large-scale deletions. Some of the structures of atrophied domains show novel protein-protein interaction interfaces that appear to compensate and stabilise their folds. Protein-protein interactions are largely influenced by the surface and charge complementarity, while RNA-RNA interactions are governed by base-pair complementarity; both interaction types are inherently different and these differences might be observed in their interaction networks. Based on this hypothesis I have explored the protein-protein, RNA-protein and the RNA-RNA interaction networks of yeast in the second chapter. By analysing the three networks I found no major differences in their network properties, which indicates an underlying uniformity in their interactomes despite their individual differences. In the third chapter I focus on RNA-protein interactions by investigating post-translational modifications (PTMs) in RNA-binding proteins (RBPs). By comparing occurrences of PTMs, I observe that RBPs significantly undergo more PTMs than non-RBPs. I also found that within RBPs, PTMs are more frequently targeted at regions that directly interact with RNA compared to regions that do not. Moreover disorderedness and amino acid composition were not observed to significantly influence the differential PTMs observed between RBPs and nonRBPs. The results point to a direct regulatory role of PTMs in RNA-protein interactions of RBPs. In the last chapter, I explore regulatory RNA-RNA interactions. Using differential expression data of mRNAs and lncRNAs from mouse models of hereditary hemochromatosis, I investigated competing regulatory interactions between mRNA, lncRNA and miRNA. A mutual interaction network was created from the predicted miRNA interaction sites on mRNAs and lncRNAs to identify regulatory RNAs in the disease. I also observed interesting relations between the sense-antisense mRNA-lncRNA pairs that indicate mutual regulation of expression levels through a yet unknown mechanism

Computational and Experimental Approaches to Reveal the Effects of Single Nucleotide Polymorphisms with Respect to Disease Diagnostics

DNA mutations are the cause of many human diseases and they are the reason for natural differences among individuals by affecting the structure, function, interactions, and other properties of DNA and expressed proteins. The ability to predict whether a given mutation is disease-causing or harmless is of great importance for the early detection of patients with a high risk of developing a particular disease and would pave the way for personalized medicine and diagnostics. Here we review existing methods and techniques to study and predict the effects of DNA mutations from three different perspectives: in silico, in vitro and in vivo. It is emphasized that the problem is complicated and successful detection of a pathogenic mutation frequently requires a combination of several methods and a knowledge of the biological phenomena associated with the corresponding macromolecules

Evolutionary, structural and functional features of cellular signalling networks

The post-translational modification of proteins is a fundamental means of biological information processing, with important functions in development, homeostasis and disease. Post-translational modifications (PTMs) can dynamically diversify the proteome in response to intracellular and extracellular signals. Since thousands of modified residues as well as entirely new modification types have recently been discovered in proteins, elucidating their biological functions and identifying the protein components of these PTM systems is a fundamental problem. Chapter 1 gives an overview of the types and known biological functions of different PTMs, as well as experimental methods used to detect them. Intrinsic disorder in proteins is introduced as a structural feature which may influence local evolutionary rates. Several examples of complex PTM signalling systems are then described. Chapter 2 presents a study of the evolution of modified amino acids in human proteins. By analysing sequence, polymorphism and mutation data at the species, population and individual levels, we observed significant evolutionary constraints on all PTM types for which extensive data was available, as well as overrepresentation of amino acids which mimic modified residues at equivalent positions. Chapter 3 applies a framework for the identification of important components of PTM signalling systems to lysine acetylation. The proteins of this system were found to be similarly conserved as essential genes. Their evolutionary histories suggested a conserved origin in chromatin regulation, followed by functional diversification. Chapter 4 extends the scope to signalling via transcriptional regulation, and presents a comprehensive overview of the interactome of the stem cell transcription factor Oct4. The results presented here facilitated characterisation of a novel post-translational modifier of Oct4, the glycosyltransferase Ogt. Chapter 5 highlights my key findings from applying evolutionary and data integration approaches to signalling networks, and outlines their implications for the study of novel signalling systems and for their engineering in synthetic biology. This dissertation therefore illuminates evolutionary, structural and functional principles of cellular signalling networks across species and within populations.Herchel Smith Research Studentshi

From Single Proteins to the Proteome : Crosslinking Mass Spectrometry - a Simple Technique with Broad Implications

Protein-protein interactions are essential for cellular processes. Detection of protein-protein interactions on both a small scale and a whole-of-cell scale is crucial to understanding life. Many technologies have been developed to detect protein-protein interactions, but few are capable of detecting these on large scale under native conditions. Crosslinking-mass spectrometry has been used for some time to study protein-protein interactions on a small-scale (single protein to protein complexes) and has the potential for application to intact cells, resulting in large protein interaction networks. There are many considerations when adapting a small-scale technology to a large-scale system. This thesis aims to investigate these and present means by which they can be addressed. Firstly, two crosslinking-mass spectrometry pipelines are used to comprehensively examine the interaction between yeast enzyme Hmt1p methyltransferase and its substrate Npl3p, and the results from the pipelines are compared. Secondly, acquisition strategies learnt from the first study are used to examine a system involving crosstalk of protein post-translational modifications (PTMs). Protein Nop1p is studied with crosslinking-mass spectrometry to detect changes in interactions between phosphorylated Nop1p and Hmt1p methyltransferase, and methylated Nop1p and Sky1p kinase. Thirdly, knowledge generated from studies one and two is applied to a large-scale sample. Protein-correlation profiling and crosslinking-mass spectrometry are coupled and used to predict novel interactions from size-exclusion fractionated yeast lysate. Together these three studies address current gaps in the crosslinking-mass spectrometry field; they assess the confidence of identifications, use reduced sample complexity for increased identifications and determine how interactions controlled by PTMs can be studied

Selectivity of the CUBAN domain in the recognition of ubiquitin and NEDD8

Among the members of the Ubiquitin‐like (Ubl) protein family, Neural precursor cell expressed developmentally down‐regulated protein 8 (NEDD8) is the closest in sequence to ubiquitin (57% identity). The two modification mechanisms and their functions, however, are highly distinct and the two Ubls are not interchangeable. A complex network of interactions between modifying enzymes and adaptors, most of which are specific while others are promiscuous, ensures selectivity. Many domains that bind the ubiquitin hydrophobic patch also bind NEDD8 while no domain that specifically binds NEDD8 has yet been described. Here we report an unbiased selection of domains that bind ubiquitin and/or NEDD8 and we characterize their specificity/promiscuity. Many ubiquitin binding domains bind ubiquitin preferentially and, to a lesser extent, NEDD8. In a few cases, the affinity of these domains for NEDD8 can be increased by substituting the alanine at position 72 with arginine, as in ubiquitin. We have also identified a unique domain, mapping to the carboxyl‐end of the protein KHNYN, which has a starkly preference for NEDD8. Given its ability to bind neddylated cullins we have named this domain CUBAN (Cullin Binding domain Associating with NEDD8). We present here the solution structure of the CUBAN domain both in the isolated form and in complex with NEDD8. The results contribute to the understanding of the discrimination mechanism between ubiquitin and the Ubl. They also provide new insights on the biological role of a ill‐defined protein, whose function is hitherto only predicted

Inference of biomolecular interactions from sequence data

This thesis describes our work on the inference of biomolecular interactions from sequence data. In particular, the first part of the thesis focuses on proteins and describes computational methods that we have developed for the inference of both intra- and inter-protein interactions from genomic data. The second part of the thesis centers around protein-RNA interactions and describes a method for the inference of binding motifs of RNA-binding proteins from high-throughput sequencing data. The thesis is organized as follows. In the first part, we start by introducing a novel mathematical model for the characterization of protein sequences (chapter 1). We then show how, using genomic data, this model can be successfully applied to two different problems, namely to the inference of interacting amino acid residues in the tertiary structure of protein domains (chapter 2) and to the prediction of protein-protein interactions in large paralogous protein families (chapters 3 and 4). We conclude the first part by a discussion of potential extensions and generalizations of the methods presented (chapter 5). In the second part of this thesis, we first give a general introduction about RNA- binding proteins (chapter 6). We then describe a novel experimental method for the genome-wide identification of target RNAs of RNA-binding proteins and show how this method can be used to infer the binding motifs of RNA-binding proteins (chapter 7). Finally, we discuss a potential mechanism by which KH domain-containing RNA- binding proteins could achieve the specificity of interaction with their target RNAs and conclude the second part of the thesis by proposing a novel type of motif finding algorithm tailored for the inference of their recognition elements (chapter 8)

Use of Protein Crosslinking and Tandem Mass Spectrometry to Study the PsbO, PsbP and PsbQ Extrinsic Proteins of Higher Plant Photosystem II

Photosystem II (PSII) is a light-driven, water plastoquinone oxidoreductase present in all oxygenic photosynthetic organisms. The oxygen evolution process is catalyzed by the Mn4CaO5 cluster and an ensemble of intrinsic and extrinsic proteins which are associated with the photosystem. This metal cluster is stabilized and protected from exogenous reductants by the extrinsic proteins, PsbO, PsbP and PsbQ in higher plants, which are present on the lumenal face of PSII. No crystal structure for the higher plant PSII is currently available; consequently, the binding locations of these extrinsic proteins in PSII remain elusive. We have used chemical-crosslinkers Bis (sulfosuccinimidyl) suberate (BS3) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to crosslink the extrinsic proteins in their bound state to PSII followed by identification of the crosslinked products by tandem mass spectrometry. BS3 crosslinking identified the interacting domain of PsbP with PsbQ involving the PsbP residues 93Y, 96K and 97T (located in the 17-residue loop 3A, 89G-105S) which are in close proximity (\u3c11.4Å) to the N-terminal 1E residue of PsbQ. We also found that this PsbP assumes a compact structure from the nine independent crosslinked residues between the N- and C-terminus of PsbP. This suggests that the N-terminus of PsbP, 1A-11K (which is not resolved in the current crystal structures), is closely associated with the C-terminal domain 170K-186A. Additionally, interacting domains of two PsbQ copies from different PSII monomers were identified. The residue pairs 98K-133Y and 101K-133Y of PsbQ were crosslinked. These residues are \u3e30 Å apart when mapped onto the PsbQ crystal structure. Since BS3 can only crosslink residues which are within 11.4 Å, these residues are hypothesized as inter-molecular crosslinks of PsbQ. Furthermore, EDC crosslinking provided structural information pertaining to the organization of the N-terminus, absent in the cyanobacterial-PsbO. In this study, twenty-four crosslinked residues located in the N-terminal, loop and the β-barrel region of PsbO were identified. The models incorporating crosslinking data suggests several differences in cyanobacterial- and higher plant-PsbO. The results on extrinsic proteins provide significant new information concerning the association of the extrinsic proteins with PSII and are valuable while proposing overall models of higher plant PSII