Nuclear Receptor Interdomain Communication is Mediated by the Hinge with Ligand Specificity.

Nuclear receptors are ligand-induced transcription factors that bind directly to target genes and regulate their expression. Ligand binding initiates conformational changes that propagate to other domains, allosterically regulating their activity. The nature of this interdomain communication in nuclear receptors is poorly understood, largely owing to the difficulty of experimentally characterizing full-length structures. We have applied computational modeling approaches to describe and study the structure of the full-length farnesoid X receptor (FXR), approximated by the DNA binding domain (DBD) and ligand binding domain (LBD) connected by the flexible hinge region. Using extended molecular dynamics simulations (>10 microseconds) and enhanced sampling simulations, we provide evidence that ligands selectively induce domain rearrangement, leading to interdomain contact. We use protein-protein interaction assays to provide experimental evidence of these interactions, identifying a critical role of the hinge in mediating interdomain contact. Our results illuminate previously unknown aspects of interdomain communication in FXR and provide a framework to enable characterization of other full-length nuclear receptors.

Gaining Insight into Large Gene Families with the Aid of Bioinformatic Tools.

Proteins participating in plant cell morphogenesis are often encoded by large gene families, in some cases comprising paralogs with variable (modular) domain organization, as in the case of the formin (FH2 protein) family of actin nucleators that can have also additional functions. Unravelling the phylogeny of such a complex gene family brings a number of specific challenges but may be crucial for predictions of protein function and for experimental design. Here we present an overview of our "cottage industry" semi-manual bioinformatic approach, based mostly, though not exclusively, on freely available software tools, which we used to obtain insight into the evolutionary history of plant FH2 proteins and some other components of the plant cell morphogenesis apparatus.

On the Effects of Disordered Tails, Supertertiary Structure and Quinary Interactions on the Folding and Function of Protein Domains.

The vast majority of our current knowledge about the biochemical and biophysical properties of proteins derives from in vitro studies conducted on isolated globular domains. However, a very large fraction of the proteins expressed in the eukaryotic cell are structurally more complex. In particular, the discovery that up to 40% of the eukaryotic proteins are intrinsically disordered, or possess intrinsically disordered regions, and are highly dynamic entities lacking a well-defined three-dimensional structure, revolutionized the structure-function paradigm and our understanding of proteins. Moreover, proteins are mostly characterized by the presence of multiple domains, influencing each other by intramolecular interactions. Furthermore, proteins exert their function in a crowded intracellular milieu, transiently interacting with a myriad of other macromolecules. In this review we summarize the literature tackling these themes from both the theoretical and experimental perspectives, highlighting the effects on protein folding and function that are played by (i) flanking disordered tails; (ii) contiguous protein domains; (iii) interactions with the cellular environment, defined as quinary structures. We show that, in many cases, both the folding and function of protein domains is remarkably perturbed by the presence of these interactions, pinpointing the importance to increase the level of complexity of the experimental work and to extend the efforts to characterize protein domains in more complex contexts.

A Folding Insulator Defines Cryptic Domains in Tropomyosin.

Multidomain proteins are the product of evolutionary selection for diversity of function through concatenation and repurposing of existing modular units of structures. In structures of proteins with multiple domains, components are often globular units stitched together with flexible linkers. Multidomain proteins often fold as multiple distinct order-disorder transitions. However, the relationship between structure and folding is not always straightforward. Tropomyosin binds to actin in muscle and cytoskeletal filaments. The structure is that of a continuous ɑ-helix lacking domain boundaries, but unfolding shows distinct transitions suggesting at least three possible domains do exist. To explore how domains might occur in a continuous structure, we used Lifson-Roig helix-coil models with sequence domains of varying helical nucleation propensities. Of these models, ones with a central folding insulator, separating folding of N- and C-terminal domains, are most consistent with experimental folding studies. The positions of domain boundaries are identified by hydrogen-deuterium exchange mass spectrometry. The presence of structurally cryptic folding domains in tropomyosin could relate to its evolution and explain the uneven distribution of deleterious mutations that lead to various cardiomyopathies.

Assessment of domain interactions in the fourteenth round of the Critical Assessment of Structure Prediction (CASP14).

The high accuracy of some CASP14 models at the domain level prompted a more detailed evaluation of structure predictions on whole targets. For the first time in critical assessment of structure prediction (CASP), we evaluated accuracy of difficult domain assembly in models submitted for multidomain targets where the community predicted individual evaluation units (EUs) with greater accuracy than full-length targets. Ten proteins with domain interactions that did not show evidence of conformational change and were not involved in significant oligomeric contacts were chosen as targets for the domain interaction assessment. Groups were ranked using complementary interaction scores (F1, QS score, and Jaccard coefficient), and their predictions were evaluated for their ability to correctly model inter-domain interfaces and overall protein folds. Target performance was broadly grouped into two clusters. The first consisted primarily of targets containing two EUs wherein predictors more broadly predicted domain positioning and interfacial contacts correctly. The other consisted of complex two-EU and three-EU targets where few predictors performed well. The highest ranked predictor, AlphaFold2, produced high-accuracy models on eight out of 10 targets. Their interdomain scores on three of these targets were significantly higher than all other groups and were responsible for their overall outperformance in the category. We further highlight the performance of AlphaFold2 and the next best group, BAKER-experimental on several interesting targets.

Periscope Proteins are variable-length regulators of bacterial cell surface interactions.

Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal "Periscope Proteins" as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus, variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly available long-read genome sequencing data, we propose that this class could contain over 50 distinct proteins, including those implicated in host colonization and biofilm formation by human pathogens. In large multidomain proteins, sequence divergence between adjacent domains appears to reduce interdomain misfolding. Periscope Proteins break this "rule," suggesting that their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria, and the immune system.

Synthetic protein scaffolds for the colocalisation of co-acting enzymes.

Nature relies on complexes of colocated enzymes to efficiently perform multiple catalytic steps. Such enzyme colocalisation promotes substrate channelling, enhances the activity of multiple synergistically acting enzymes and avoids the loss of potentially toxic intermediates. The industrial biotechnology field develops sophisticated methods to mimic natural colocalisation mechanisms to produce increasingly complex bio-based chemicals. Synthetic protein scaffolds are an advanced way to achieve colocalisation of multiple enzymes in one protein complex. The backbone scaffold is composed of multiple domains that are either separated by linkers or fused to self-assembling proteins. Enzymes are recruited to this scaffold by fusing them to domains that bind to orthogonal domains in the scaffold. A particular feature of synthetic protein scaffolds is the control over spatial organisation and enzyme stoichiometry. Several successful examples of synthetic protein scaffolds have been reported, yet the optimisation of such multi-enzyme complexes is a multiparametric, and therefore often empirical process. This review focusses on pioneering scaffolding examples and elaborates on each parameter influencing the activity of these multi-enzyme complexes. Advances in this field are expected to result in a growing catalogue of chemicals that can be produced starting from cheap and widely available renewable materials.

Multiple Forms of Multifunctional Proteins in Health and Disease.

Protein science has moved from a focus on individual molecules to an integrated perspective in which proteins emerge as dynamic players with multiple functions, rather than monofunctional specialists. Annotation of the full functional repertoire of proteins has impacted the fields of biochemistry and genetics, and will continue to influence basic and applied science questions - from the genotype-to-phenotype problem, to our understanding of human pathologies and drug design. In this review, we address the phenomena of pleiotropy, multidomain proteins, promiscuity, and protein moonlighting, providing examples of multitasking biomolecules that underlie specific mechanisms of human disease. In doing so, we place in context different types of multifunctional proteins, highlighting useful attributes for their systematic definition and classification in future research directions.

Successes and challenges in simulating the folding of large proteins.

Computational simulations of protein folding can be used to interpret experimental folding results, to design new folding experiments, and to test the effects of mutations and small molecules on folding. However, whereas major experimental and computational progress has been made in understanding how small proteins fold, research on larger, multidomain proteins, which comprise the majority of proteins, is less advanced. Specifically, large proteins often fold via long-lived partially folded intermediates, whose structures, potentially toxic oligomerization, and interactions with cellular chaperones remain poorly understood. Molecular dynamics based folding simulations that rely on knowledge of the native structure can provide critical, detailed information on folding free energy landscapes, intermediates, and pathways. Further, increases in computational power and methodological advances have made folding simulations of large proteins practical and valuable. Here, using serpins that inhibit proteases as an example, we review native-centric methods for simulating the folding of large proteins. These synergistic approaches range from Go and related structure-based models that can predict the effects of the native structure on folding to all-atom-based methods that include side-chain chemistry and can predict how disease-associated mutations may impact folding. The application of these computational approaches to serpins and other large proteins highlights the successes and limitations of current computational methods and underscores how computational results can be used to inform experiments. These powerful simulation approaches in combination with experiments can provide unique insights into how large proteins fold and misfold, expanding our ability to predict and manipulate protein folding.

New Perspectives on Plant Adenylyl Cyclases.

It is increasingly clear that plant genomes encode numerous complex multidomain proteins that harbor functional adenylyl cyclase (AC) centers. These AC containing proteins have well-documented roles in development and responses to the environment. However, it is only for a few of these proteins that we are beginning to understand the intramolecular mechanisms that govern their cellular and biological functions, as detailed characterizations are biochemically and structurally challenging given that these poorly conserved AC centers typically constitute only a small fraction (<10%) of complex plant proteins. Here, we offer fresh perspectives on their seemingly cryptic activities specifically showing evidence for the presence of multiple functional AC centers in a single protein and linking their catalytic strengths to the Mg2+/Mn2+-binding amino acids. We used a previously described computational approach to identify candidate multidomain proteins from Arabidopsis thaliana that contain multiple AC centers and show, using an Arabidopsis leucine-rich repeat containing protein (TAIR ID: At3g14460; AtLRRAC1) as example, biochemical evidence for multienzymatic activities. Importantly, all AC-containing fragments of this protein can complement the AC-deficient mutant cyaA in Escherichia coli, while structural modeling coupled with molecular docking simulations supports catalytic feasibility albeit to varying degrees as determined by the frequency of suitable substrate binding poses predicted for the AC sites. This statistic correlates well with the enzymatic assays, which implied that the greatly reduced AC activities is due to the absence of the negatively charged [DE] amino acids previously assigned to cation-, in particular Mg2+/Mn2+-binding roles in ACs.

Energetic dependencies dictate folding mechanism in a complex protein.

Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.

Specialized structural and functional roles of residues selectively conserved in subfamilies of the pleckstrin homology domain family.

Homologous domains embedded in multidomain proteins of different domain architectures (DA) may exhibit subtle, but important, differences in their structure and function. Here, we consider two multidomain proteins, Arf nucleotide binding site opener (ARNO) and G protein-coupled receptor kinase 2 (GRK2), which have very different DAs, but both contain pleckstrin homology (PH) domains. We analyzed the roles of residues selectively conserved in these subfamilies of PH domains from ARNO and GRK2 proteins. DA-specific residues in PH domain are found to contribute to structural and functional specialization of ARNO and GRK2 in terms of (a) specific intra- and interprotein interactions; (b) specificity for phospholipids; and (c) participation in conformational excursions, leading to various functional forms. Our approach can also be applied to subfamilies of other protein families to identify subfamily-specific residues and their specialized roles.

Eukaryote specific folds: Part of the whole.

The origin of eukaryotes is one of the central transitions in the history of life; without eukaryotes there would be no complex multicellular life. The most accepted scenarios suggest the endosymbiosis of a mitochondrial ancestor with a complex archaeon, even though the details regarding the host and the triggering factors are still being discussed. Accordingly, phylogenetic analyses have demonstrated archaeal affiliations with key informational systems, while metabolic genes are often related to bacteria, mostly to the mitochondrial ancestor. Despite of this, there exists a large number of protein families and folds found only in eukaryotes. In this study, we have analyzed structural superfamilies and folds that probably appeared during eukaryogenesis. These folds typically represent relatively small binding domains of larger multidomain proteins. They are commonly involved in biological processes that are particularly complex in eukaryotes, such as signaling, trafficking/cytoskeleton, ubiquitination, transcription and RNA processing, but according to recent studies, these processes also have prokaryotic roots. Thus the folds originating from an eukaryotic stem seem to represent accessory parts that have contributed in the expansion of several prokaryotic processes to a new level of complexity. This might have taken place as a co-evolutionary process where increasing complexity and fold innovations have supported each other.

Correction: The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength.

[This corrects the article on p. 755 in vol. 8, PMID: 29163152.].

Modification of the kinetic stability of immunoglobulin G by solvent additives.

Biophysical properties of antibody-based biopharmaceuticals are a critical part of their release criteria. In this context, finding the appropriate formulation is equally important as optimizing their intrinsic biophysical properties through protein engineering, and both are mutually dependent. Most previous studies have empirically tested the impact of additives on measures of colloidal stability, while mechanistic aspects have usually been limited to only the thermodynamic stability of the protein. Here we emphasize the kinetic impact of additives on the irreversible denaturation steps of immunoglobulins G (IgG) and their antigen-binding fragments (Fabs), as these are the key committed steps preceding aggregation, and thus especially informative in elucidating the molecular parameters of activity loss. We examined the effects of ten additives on the conformational kinetic stability by differential scanning calorimetry (DSC), using a recently developed three-step model containing both reversible and irreversible steps. The data highlight and help to rationalize different effects of the additives on the properties of full-length IgG, analyzed by onset and aggregation temperatures as well as by kinetic parameters derived from our model. Our results further help to explain the observation that stabilizing mutations in the antigen-binding fragment (Fab) significantly affect the kinetic parameters of its thermal denaturation, but not the aggregation properties of the full-length IgGs. We show that the proper analysis of DSC scans for full-length IgGs and their corresponding Fabs not only helps in ranking their stability in different formats and formulations, but provides important mechanistic insights for improving the conformational kinetic stability of IgGs.

The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength.

NADPH-cytochrome P450 reductase (CPR) is a redox partner of microsomal cytochromes P450 and is a prototype of the diflavin reductase family. CPR contains 3 distinct functional domains: a FMN-binding domain (acceptor reduction), a linker (hinge), and a connecting/FAD domain (NADPH oxidation). It has been demonstrated that the mechanism of CPR exhibits an important step in which it switches from a compact, closed conformation (locked state) to an ensemble of open conformations (unlocked state), the latter enabling electron transfer to redox partners. The conformational equilibrium between the locked and unlocked states has been shown to be highly dependent on ionic strength, reinforcing the hypothesis of the presence of critical salt interactions at the interface between the FMN and connecting FAD domains. Here we show that specific residues of the hinge segment are important in the control of the conformational equilibrium of CPR. We constructed six single mutants and two double mutants of the human CPR, targeting residues G240, S243, I245 and R246 of the hinge segment, with the aim of modifying the flexibility or the potential ionic interactions of the hinge segment. We measured the reduction of cytochrome c at various salt concentrations of these 8 mutants, either in the soluble or membrane-bound form of human CPR. All mutants were found capable of reducing cytochrome c yet with different efficiency and their maximal rates of cytochrome c reduction were shifted to lower salt concentration. In particular, residue R246 seems to play a key role in a salt bridge network present at the interface of the hinge and the connecting domain. Interestingly, the effects of mutations, although similar, demonstrated specific differences when present in the soluble or membrane-bound context. Our results demonstrate that the electrostatic and flexibility properties of the hinge segment are critical for electron transfer from CPR to its redox partners.

Effects of Fluorophore Attachment on Protein Conformation and Dynamics Studied by spFRET and NMR Spectroscopy.

Fluorescence-based techniques are widely used to study biomolecular conformations, intra- and intermolecular interactions, and conformational dynamics of macromolecules. Especially for fluorescence-based single-molecule experiments, the choice of the fluorophore and labeling position are highly important. In this work, we studied the biophysical and structural effects that are associated with the conjugation of fluorophores to cysteines in the splicing factor U2AF65 by using single pair Förster resonance energy transfer (FRET) and nuclear magnetic resonance (NMR) spectroscopy. It is shown that certain acceptor fluorophores are advantageous depending on the experiments performed. The effects of dye attachment on the protein conformation were characterized using heteronuclear NMR experiments. The presence of hydrophobic and aromatic moieties in the fluorophores can significantly affect the conformation of the conjugated protein, presumably by transient interactions with the protein surface. Guidelines are provided for carefully choosing fluorophores, considering their photophysical properties and chemical features for the design of FRET experiments, and for minimizing artifacts.

Regulated methionine oxidation by monooxygenases.

Protein function can be regulated via post-translational modifications by numerous enzymatic and non-enzymatic mechanisms, including oxidation of cysteine and methionine residues. Redox-dependent regulatory mechanisms have been identified for nearly every cellular process, but the major paradigm has been that cellular components are oxidized (damaged) by reactive oxygen species (ROS) in a relatively unspecific way, and then reduced (repaired) by designated reductases. While this scheme may work with cysteine, it cannot be ascribed to other residues, such as methionine, whose reaction with ROS is too slow to be biologically relevant. However, methionine is clearly oxidized in vivo and enzymes for its stereoselective reduction are present in all three domains of life. Here, we revisit the chemistry and biology of methionine oxidation, with emphasis on its generation by enzymes from the monooxygenase family. Particular attention is placed on MICALs, a recently discovered family of proteins that harbor an unusual flavin-monooxygenase domain with an NADPH-dependent methionine sulfoxidase activity. Based on structural and kinetic information we provide a rational framework to explain MICAL mechanism, inhibition, and regulation. Methionine residues that are targeted by MICALs are reduced back by methionine sulfoxide reductases, suggesting that reversible methionine oxidation may be a general mechanism analogous to the regulation by phosphorylation by kinases/phosphatases. The identification of new enzymes that catalyze the oxidation of methionine will open a new area of research at the forefront of redox signaling.

Visualizing Evolutionary Relationships of Multidomain Proteins: An Example from Receiver (REC) Domains of Sensor Histidine Kinases in the Candidatus Maribeggiatoa str. Orange Guaymas Draft Genome.

For multidomain proteins, evolutionary changes may occur at the domain as well as the whole-protein level. An example is presented here, with suggestions for how such complicated relationships might be visualized. Earlier analysis of the Candidatus Maribeggiatoa str. Orange Guaymas (BOGUAY; Gammaproteobacteria) single-filament draft genome found evidence of gene exchange with the phylogenetically distant Cyanobacteria, particularly for sensory and signal transduction proteins. Because these are modular proteins, known to undergo frequent duplication, domain swapping, and horizontal gene transfer, a single domain was chosen for analysis. Recognition (REC) domains are short (~125 amino acids) and well conserved, simplifying sequence alignments and phylogenetic calculations. Over 100 of these were identified in the BOGUAY genome and found to have a wide range of inferred phylogenetic relationships. Two sets were chosen here for detailed study. One set of four BOGUAY ORFs has closest relatives among other Beggiatoaceae and Cyanobacteria. A second set of four has REC domains with more mixed affiliations, including other Beggiatoaceae, several sulfate-reducing Deltaproteobacteria and Firmicutes, magnetotactic Nitrospirae, one Shewanella and one Ferrimonas strain (both Gammaproteobacteria), and numerous Vibrio vulnificus and V. navarrensis strains (also Gammaproteobacteria). For an overview of the possible origins of the whole proteins and the surrounding genomic regions, color-coded BLASTP results were produced and displayed against cartoons showing protein domain structure of predicted genes. This is suggested as a visualization method for investigation of possible horizontally transferred regions, giving more detail than scans of DNA composition and codon usage but much faster than carrying out full phylogenetic analyses for multiple proteins. As expected, most of the predicted sensor histidine kinases investigated have two or more segments with distinct BLASTP affiliations. For the first set of BOGUAY ORFs, the flanking regions were also examined, and the results suggest they are embedded in genomic stretches with complex histories. An automated method of creating such visualizations could be generally useful; a wish list for its features is given.

Application of Nuclear Magnetic Resonance and Hybrid Methods to Structure Determination of Complex Systems.

The current main challenge of Structural Biology is to undertake the structure determination of increasingly complex systems in the attempt to better understand their biological function. As systems become more challenging, however, there is an increasing demand for the parallel use of more than one independent technique to allow pushing the frontiers of structure determination and, at the same time, obtaining independent structural validation. The combination of different Structural Biology methods has been named hybrid approaches. The aim of this review is to critically discuss the most recent examples and new developments that have allowed structure determination or experimentally-based modelling of various molecular complexes selecting them among those that combine the use of nuclear magnetic resonance and small angle scattering techniques. We provide a selective but focused account of some of the most exciting recent approaches and discuss their possible further developments.