The Role of Low Complexity Regions in Protein Interaction Modes: An Illustration in Huntingtin

Low complexity regions (LCRs) are very frequent in protein sequences, generally having a lower propensity to form structured domains and tending to be much less evolutionarily conserved than globular domains. Their higher abundance in eukaryotes and in species with more cellular types agrees with a growing number of reports on their function in protein interactions regulated by post-translational modifications. LCRs facilitate the increase of regulatory and network complexity required with the emergence of organisms with more complex tissue distribution and development. Although the low conservation and structural flexibility of LCRs complicate their study, evolutionary studies of proteins across species have been used to evaluate their significance and function. To investigate how to apply this evolutionary approach to the study of LCR function in protein–protein interactions, we performed a detailed analysis for Huntingtin (HTT), a large protein that is a hub for interaction with hundreds of proteins, has a variety of LCRs, and for which partial structural information (in complex with HAP40) is available. We hypothesize that proteins RASA1, SYN2, and KAT2B may compete with HAP40 for their attachment to the core of HTT using similar LCRs. Our results illustrate how evolution might favor the interplay of LCRs with domains, and the possibility of detecting multiple modes of LCR-mediated protein–protein interactions with a large hub such as HTT when enough protein interaction data is available.     

VDACs Post-Translational Modifications Discovery by Mass Spectrometry: Impact on Their Hub Function

VDAC (voltage-dependent anion selective channel) proteins, also known as mitochondrial porins, are the most abundant proteins of the outer mitochondrial membrane (OMM), where they play a vital role in various cellular processes, in the regulation of metabolism, and in survival pathways. There is increasing consensus about their function as a cellular hub, connecting bioenergetics functions to the rest of the cell. The structural characterization of VDACs presents challenging issues due to their very high hydrophobicity, low solubility, the difficulty to separate them from other mitochondrial proteins of similar hydrophobicity and the practical impossibility to isolate each single isoform. Consequently, it is necessary to analyze them as components of a relatively complex mixture. Due to the experimental difficulties in their structural characterization, post-translational modifications (PTMs) of VDAC proteins represent a little explored field. Only in recent years, the increasing number of tools aimed at identifying and quantifying PTMs has allowed to increase our knowledge in this field and in the mechanisms that regulate functions and interactions of mitochondrial porins. In particular, the development of nano-reversed phase ultra-high performance liquid chromatography (nanoRP-UHPLC) and ultra-sensitive high-resolution mass spectrometry (HRMS) methods has played a key role in this field. The findings obtained on VDAC PTMs using such methodologies, which permitted an in-depth characterization of these very hydrophobic trans-membrane pore proteins, are summarized in this review.     

Experimental and Computational Characterization of Biological Liquid Crystals: A Review of Single-Molecule Bioassays

Quantitative understanding of the mechanical behavior of biological liquid crystals such as proteins is essential for gaining insight into their biological functions, since some proteins perform notable mechanical functions. Recently, single-molecule experiments have allowed not only the quantitative characterization of the mechanical behavior of proteins such as protein unfolding mechanics, but also the exploration of the free energy landscape for protein folding. In this work, we have reviewed the current state-of-art in single-molecule bioassays that enable quantitative studies on protein unfolding mechanics and/or various molecular interactions. Specifically, single-molecule pulling experiments based on atomic force microscopy (AFM) have been overviewed. In addition, the computational simulations on single-molecule pulling experiments have been reviewed. We have also reviewed the AFM cantilever-based bioassay that provides insight into various molecular interactions. Our review highlights the AFM-based single-molecule bioassay for quantitative characterization of biological liquid crystals such as proteins.     

SARS-CoV-2 Membrane Protein: From Genomic Data to Structural New Insights

Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) is composed of four structural proteins and several accessory non-structural proteins. SARS-CoV-2’s most abundant structural protein, Membrane (M) protein, has a pivotal role both during viral infection cycle and host interferon antagonism. This is a highly conserved viral protein, thus an interesting and suitable target for drug discovery. In this paper, we explain the structural nature of M protein homodimer. To do so, we developed and applied a detailed and robust in silico workflow to predict M protein dimeric structure, membrane orientation, and interface characterization. Single Nucleotide Polymorphisms (SNPs) in M protein were retrieved from over 1.2 M SARS-CoV-2 genomes and proteins from the Global Initiative on Sharing All Influenza Data (GISAID) database, 91 of which were located at the predicted dimer interface. Among those, we identified SNPs in Variants of Concern (VOC) and Variants of Interest (VOI). Binding free energy differences were evaluated for dimer interfacial SNPs to infer mutant protein stabilities. A few high-prevalent mutated residues were found to be especially relevant in VOC and VOI. This realization may be a game-changer to structure-driven formulation of new therapeutics for SARS-CoV-2.     

Protein Crystallography in Vaccine Research and Development

The use of protein X-ray crystallography for structure-based design of small-molecule drugs is well-documented and includes several notable success stories. However, it is less well-known that structural biology has emerged as a major tool for the design of novel vaccine antigens. Here, we review the important contributions that protein crystallography has made so far to vaccine research and development. We discuss several examples of the crystallographic characterization of vaccine antigen structures, alone or in complexes with ligands or receptors. We cover the critical role of high-resolution epitope mapping by reviewing structures of complexes between antigens and their cognate neutralizing, or protective, antibody fragments. Most importantly, we provide recent examples where structural insights obtained via protein crystallography have been used to design novel optimized vaccine antigens. This review aims to illustrate the value of protein crystallography in the emerging discipline of structural vaccinology and its impact on the rational design of vaccines.     

Elongation Factors EFIA and EF-Tu: Their Role in Translation and Beyond

Protein biosynthesis is a central process in cell life. Although the ribosome provides the machinery for biosynthesis, successful translation of the information contained in mRNA also relies on a number of protein factors that transiently associate with this giant ribonucleic complex. Among them, the elongation factors EF-Tu (bacteria) and EFIA (eukarya and archaea), which carry the aa-tRNA to the ribosome, have earned a special consideration. Here we present an excursus of structural information collected in the last decade on these enzymes. A significant advance in this field has been the recent crystallographic characterization of the complex between ribosome and EF-Tu. These findings, along with structural studies performed on EF-Tu complexes with small molecules and proteins, have provided a picture of the interactions of these proteins with a differentiate variety of biological partners. In contrast to EF-Tu, t he structural characterization of the states assumed by archaeal and eukaryal EFIA is largely incomplete. Here we also review solution and computational studies that have been successfully applied to integrate and complement crystallographic data. Analogies and differences between EFs isolated from different organisms will be highlighted. The new challenges of this research area, including the structural interpretation of the role of these proteins in other biological processes, are also addressed.     

Adenovirus Structure: What Is New?

Adenoviruses are large (~950 Å) and complex non-enveloped, dsDNA icosahedral viruses. They have a pseudo-T = 25 triangulation number with at least 12 different proteins composing the virion. These include the major and minor capsid proteins, core proteins, maturation protease, terminal protein, and packaging machinery. Although adenoviruses have been studied for more than 60 years, deciphering their architecture has presented a challenge for structural biology techniques. An outstanding event was the first near-atomic resolution structure of human adenovirus type 5 (HAdV-C5), solved by cryo-electron microscopy (cryo-EM) in 2010. Discovery of new adenovirus types, together with methodological advances in structural biology techniques, in particular cryo-EM, has lately produced a considerable amount of new, high-resolution data on the organization of adenoviruses belonging to different species. In spite of these advances, the organization of the non-icosahedral core is still a great unknown. Nevertheless, alternative techniques such as atomic force microscopy (AFM) are providing interesting glimpses on the role of the core proteins in genome condensation and virion stability. Here we summarize the current knowledge on adenovirus structure, with an emphasis on high-resolution structures obtained since 2010.     

Fake It ‘Till You Make It—The Pursuit of Suitable Membrane Mimetics for Membrane Protein Biophysics

Membrane proteins evolved to reside in the hydrophobic lipid bilayers of cellular membranes. Therefore, membrane proteins bridge the different aqueous compartments separated by the membrane, and furthermore, dynamically interact with their surrounding lipid environment. The latter not only stabilizes membrane proteins, but directly impacts their folding, structure and function. In order to be characterized with biophysical and structural biological methods, membrane proteins are typically extracted and subsequently purified from their native lipid environment. This approach requires that lipid membranes are replaced by suitable surrogates, which ideally closely mimic the native bilayer, in order to maintain the membrane proteins structural and functional integrity. In this review, we survey the currently available membrane mimetic environments ranging from detergent micelles to bicelles, nanodiscs, lipidic-cubic phase (LCP), liposomes, and polymersomes. We discuss their respective advantages and disadvantages as well as their suitability for downstream biophysical and structural characterization. Finally, we take a look at ongoing methodological developments, which aim for direct in-situ characterization of membrane proteins within native membranes instead of relying on membrane mimetics.     

Label-Free Surface-Enhanced Raman Spectroscopic Analysis of Proteins: Advances and Applications

Surface-enhanced Raman spectroscopy (SERS) is powerful for structural characterization of biomolecules under physiological condition. Owing to its high sensitivity and selectivity, SERS is useful for probing intrinsic structural information of proteins and is attracting increasing attention in biophysics, bioanalytical chemistry, and biomedicine. This review starts with a brief introduction of SERS theories and SERS methodology of protein structural characterization. SERS-active materials, related synthetic approaches, and strategies for protein-material assemblies are outlined and discussed, followed by detailed discussion of SERS spectroscopy of proteins with and without cofactors. Recent applications and advances of protein SERS in biomarker detection, cell analysis, and pathogen discrimination are then highlighted, and the spectral reproducibility and limitations are critically discussed. The review ends with a conclusion and a discussion of current challenges and perspectives of promising directions.     

Predicting residue solvent accessibility from protein sequence by considering the sequence environment

The solvent accessibility of each residue is predicted on thebasis of the protein sequence. A set of 338 monomeric, non-homologousand high-resolution protein crystal structures is used as alearning set and a jackknife procedure is applied to each entry.The prediction is based on the comparison of the observed andthe average values of the solvent-accessible area. It appearsthat the prediction accuracy is significantly improved by consideringthe residue types preceding and/or following the residue whoseaccessibility must be predicted. In contrast, the separate treatmentof different secondary structural types does not improve thequality of the prediction. It is furthermore shown that theresidue accessibility is much better predicted in small thanin larger proteins. Such a discrepancy must be carefully consideredin any algorithm for predicting residue accessibility.     

Exploring the early steps of amyloid peptide aggregation by computers

The assembly of normally soluble proteins into amyloid fibrils is a hallmark of neurodegenerative diseases. Because protein aggregation is very complex, involving a variety of oligomeric metastable intermediates, the detailed aggregation paths and structural characterization of the intermediates remain to be determined. Yet, there is strong evidence that these oligomers, which form early in the process of fibrillogenesis, are cytotoxic. In this paper, we review our current understanding of the underlying factors that promote the aggregation of peptides into amyloid fibrils. We focus here on the structural and dynamic aspects of the aggregation as observed in state-of-the-art computer simulations of amyloid-forming peptides with an emphasis on the activation-relaxation technique.     

PV1 Protein from Plasmodium falciparum Exhibits Chaperone-Like Functions and Cooperates with Hsp100s

Plasmodium falciparum parasitophorous vacuolar protein 1 (PfPV1), a protein unique to malaria parasites, is localized in the parasitophorous vacuolar (PV) and is essential for parasite growth. Previous studies suggested that PfPV1 cooperates with the Plasmodium translocon of exported proteins (PTEX) complex to export various proteins from the PV. However, the structure and function of PfPV1 have not been determined in detail. In this study, we undertook the expression, purification, and characterization of PfPV1. The tetramer appears to be the structural unit of PfPV1. The activity of PfPV1 appears to be similar to that of molecular chaperones, and it may interact with various proteins. PfPV1 could substitute CtHsp40 in the CtHsp104, CtHsp70, and CtHsp40 protein disaggregation systems. Based on these results, we propose a model in which PfPV1 captures various PV proteins and delivers them to PTEX through a specific interaction with HSP101.     

Molecular models of the interface between anterior pharynx-defective protein 1 (APH-1) and presenilin involving GxxxG motifs

Gamma-secretase is an integral membrane protease, which is a complex of four membrane proteins. Improper functioning of gamma-secretase was found to be critical in the pathogenesis of Alzheimer's disease. Despite numerous efforts, the structure of the protease as well as its proteolytic mechanism remains poorly understood. In this work we constructed a model of interactions between two proteins forming gamma-secretase: APH-1 and presenilin. This interface is based on a highly conserved GxxxGxxxG motif in the APH-1 protein. It can form a tight contact with a small-residue AxxxAxxxG motif in presenilin. Here, four binding modes based on similar structures involving GxxxG motifs in glycophorin and aquaporin were proposed and verified. The resulting best model employs antiparallel orientations of interacting helices and is in agreement with the currently accepted topology of both proteins. This model can be used for further structural characterization of gamma-secretase and its components.     

Artificially Linked Ubiquitin Dimers Characterised Structurally and Dynamically by NMR Spectroscopy

As one of the most prevalent post-translational modifications in eukaryotic cells, ubiquitylation plays vital roles in many cellular processes, such as protein degradation, DNA metabolism, and cell differentiation. Substrate proteins can be tagged by distinct types of polymeric ubiquitin (Ub) chains, which determine the eventual fate of the modified protein. A facile, click chemistry based approach for the efficient generation of linkage-defined Ub chains, including Ub dimers, was recently established. Within these chains, individual Ub moieties are connected through a triazole linkage, rather than the natural isopeptide bond. Herein, it is reported that the conformation of an artificially K48-linked Ub dimer resembles that of the natively linked dimer, with respect to structural and dynamic characteristics, as demonstrated by means of high-resolution NMR spectroscopy. Thus, it is proposed that artificially linked Ub dimers, as generated by this approach, represent potent tools for studying the inherently different properties and functions of distinct Ub chains.     

Structural genomics of proteins involved in copper homeostasis

Genome sequencing projects have provided a wealth of data, most notably the primary sequences of all the proteins that a given organism can produce. The understanding of this information at the functional level is still in the beginning stages. Three-dimensional structural information is necessary to unravel at the atomic level the mechanisms by which a protein carries out its function, and such information can often be very useful to predict at least gross functional features, even in the absence of biochemical data. An exhaustive structural characterization of the proteins encoded in the genomes is thus highly desirable. To enhance the functional insights provided by genome-scale structural determination, we have prioritized our research to target specific processes of the cell, i.e., those responsible for controlling metal homeostasis. In this Account, we present the results obtained by the Magnetic Resonance Center of the University of Florence on proteins involved in the homeostasis of copper. The general research strategy is presented, followed by a discussion focused on different key experimental aspects. An overview of the initial results and of their relevance to the understanding of molecular function and cellular processes is also given.     

Methods of studying functional characteristics of vegetable proteins

In a functional characterization, a set of data is obtained which gives information on the fields of application for a certain protein ingredient. This fingerprint can be used as a guideline in product development and restricts the amount of tests on large scale. The function of a protein in a complex food system can be better understood if a functional characterization is combined with a microstructural investigation. This may be especially important in cases where proteins act by indirect functionality, i.e., when the prescence of one protein changes the function of another component. In order to improve the efficiency of functional characterizations, methodologies need to be further developed. This is especially important for characterization of gels, emulsions, and mixtures of structures.     

Recognizing misfolded and distorted protein structures by the assumption-based similarity score

A new similarity score (-score) is proposed which is able tofind the correct protein structure among the very close alternativesand to distinguish between correct and deliberately misfoldedstructures. This score is based on the general principle `similarlikes similar', and it favors hydrophobic and hydrophilic contacts,and disfavors hydrophobic-to-hydrophilic contacts in proteins.The values of -scores calculated for the high-resolution proteinstructures from the representative set are compared with thoseof alternatives: (i) very close alternatives which are onlyslightly distorted by conformational energy minimization invacuo; (ii) alternatives with subsequently growing distortions,generated by molecular dynamics simulations in vacuo; (iii)structures derived by molecular dynamics simulation in solventat 300 K; (iv) deliberately misfolded protein models. In nearlyall tested cases the similarity score can successfully distinguishbetween experimental structure and its alternatives, even ifthe root mean square displacement of all heavy atoms is lessthan 1 Å. The confidence interval of the similarity scorewas estimated using the high-resolution X-ray structures ofdomain pairs related by non-crystallographic symmetry. The similarityscore can be used for the evaluation of the general qualityof the protein models, choosing the correct structures amongthe very close alternatives, characterization of models simulatingfolding/unfolding, etc.