Assembly Mechanism of Mucin and von Willebrand Factor Polymers.

The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here, we present an integrated structural analysis of the intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi apparatus. Remarkably, a densely O-glycosylated mucin domain performs an organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation.

Structure, dynamics, assembly, and evolution of protein complexes.

The assembly of individual proteins into functional complexes is fundamental to nearly all biological processes. In recent decades, many thousands of homomeric and heteromeric protein complex structures have been determined, greatly improving our understanding of the fundamental principles that control symmetric and asymmetric quaternary structure organization. Furthermore, our conception of protein complexes has moved beyond static representations to include dynamic aspects of quaternary structure, including conformational changes upon binding, multistep ordered assembly pathways, and structural fluctuations occurring within fully assembled complexes. Finally, major advances have been made in our understanding of protein complex evolution, both in reconstructing evolutionary histories of specific complexes and in elucidating general mechanisms that explain how quaternary structure tends to evolve. The evolution of quaternary structure occurs via changes in self-assembly state or through the gain or loss of protein subunits, and these processes can be driven by both adaptive and nonadaptive influences.

Substrate-dependent effects of quaternary structure on RNase E activity.

RNase E is an essential, multifunctional ribonuclease encoded in E. coli by the rne gene. Structural analysis indicates that the ribonucleolytic activity of this enzyme is conferred by rne-encoded polypeptide chains that (1) dimerize to form a catalytic site at the protein-protein interface, and (2) multimerize further to generate a tetrameric quaternary structure consisting of two dimerized Rne-peptide chains. We identify here a mutation in the Rne protein's catalytic region (E429G), as well as a bacterial cell wall peptidoglycan hydrolase (Amidase C [AmiC]), that selectively affect the specific activity of the RNase E enzyme on long RNA substrates, but not on short synthetic oligonucleotides, by enhancing enzyme multimerization. Unlike the increase in specific activity that accompanies concentration-induced multimerization, enhanced multimerization associated with either the E429G mutation or interaction of the Rne protein with AmiC is independent of the substrate's 5' terminus phosphorylation state. Our findings reveal a previously unsuspected substrate length-dependent regulatory role for RNase E quaternary structure and identify cis-acting and trans-acting factors that mediate such regulation.

Democratizing protein language models with parameter-efficient fine-tuning.

Proteomics has been revolutionized by large protein language models (PLMs), which learn unsupervised representations from large corpora of sequences. These models are typically fine-tuned in a supervised setting to adapt the model to specific downstream tasks. However, the computational and memory footprint of fine-tuning (FT) large PLMs presents a barrier for many research groups with limited computational resources. Natural language processing has seen a similar explosion in the size of models, where these challenges have been addressed by methods for parameter-efficient fine-tuning (PEFT). In this work, we introduce this paradigm to proteomics through leveraging the parameter-efficient method LoRA and training new models for two important tasks: predicting protein-protein interactions (PPIs) and predicting the symmetry of homooligomer quaternary structures. We show that these approaches are competitive with traditional FT while requiring reduced memory and substantially fewer parameters. We additionally show that for the PPI prediction task, training only the classification head also remains competitive with full FT, using five orders of magnitude fewer parameters, and that each of these methods outperform state-of-the-art PPI prediction methods with substantially reduced compute. We further perform a comprehensive evaluation of the hyperparameter space, demonstrate that PEFT of PLMs is robust to variations in these hyperparameters, and elucidate where best practices for PEFT in proteomics differ from those in natural language processing. All our model adaptation and evaluation code is available open-source at https://github.com/microsoft/peft_proteomics. Thus, we provide a blueprint to democratize the power of PLM adaptation to groups with limited computational resources.

Understanding a protein fold: The physics, chemistry, and biology of α-helical coiled coils.

Protein science is being transformed by powerful computational methods for structure prediction and design: AlphaFold2 can predict many natural protein structures from sequence, and other AI methods are enabling the de novo design of new structures. This raises a question: how much do we understand the underlying sequence-to-structure/function relationships being captured by these methods? This perspective presents our current understanding of one class of protein assembly, the α-helical coiled coils. At first sight, these are straightforward: sequence repeats of hydrophobic (h) and polar (p) residues, (hpphppp)n, direct the folding and assembly of amphipathic α helices into bundles. However, many different bundles are possible: they can have two or more helices (different oligomers); the helices can have parallel, antiparallel, or mixed arrangements (different topologies); and the helical sequences can be the same (homomers) or different (heteromers). Thus, sequence-to-structure relationships must be present within the hpphppp repeats to distinguish these states. I discuss the current understanding of this problem at three levels: first, physics gives a parametric framework to generate the many possible coiled-coil backbone structures. Second, chemistry provides a means to explore and deliver sequence-to-structure relationships. Third, biology shows how coiled coils are adapted and functionalized in nature, inspiring applications of coiled coils in synthetic biology. I argue that the chemistry is largely understood; the physics is partly solved, though the considerable challenge of predicting even relative stabilities of different coiled-coil states remains; but there is much more to explore in the biology and synthetic biology of coiled coils.

Experimental modification in thermal stability of oligomers by alanine substitution and site saturation mutagenesis of interfacial residues.

For certain industrial applications, the stability of protein oligomers is important. In this study, we demonstrated an efficient method to improve the thermal stability of oligomers using the trimeric protein chloramphenicol acetyltransferase (CAT) as the model. We substituted all interfacial residues of CAT with alanine to detect residues critical for oligomer stability. Mutation of six of the forty-nine interfacial residues enhanced oligomer thermal stability. Site saturation mutagenesis was performed on these six residues to optimize the side chains. About 15% of mutations enhanced thermal stability by more than 0.5 °C and most did not disrupt activity of CAT. Certain combinations of mutations further improved thermal stability and resistance against heat treatment. The quadruple mutant, H17V/N34S/F134A/D157C, retained the same activity as the wild-type after heat treatment at 9 °C higher temperature than the wild-type CAT. Furthermore, combinations with only alanine substitutions also improved thermal stability, suggesting the method we developed can be used for rapid modification of industrially important proteins.

Implementation of FRET Spectrometry Using Temporally Resolved Fluorescence: A Feasibility Study.

Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.

Diversity of Quaternary Structures Regulates Nuclear Receptor Activities.

Nuclear receptors (NRs) form homo- and/or heterodimers as central scaffolds of multiprotein complexes, which activate or repress gene transcription to regulate development, homeostasis, and metabolism. Recent studies on NR quaternary structure reveal novel mechanisms of receptor dimerization, the existence of tetrameric chromatin-bound NRs, and previously unanticipated protein-protein/protein-DNA interactions.

Biochemical, structural and dynamical characterizations of the lactate dehydrogenase from Selenomonas ruminantium provide information about an intermediate evolutionary step prior to complete allosteric regulation acquisition in the super family of lactate and malate dehydrogenases.

In this work, we investigated the lactate dehydrogenase (LDH) from Selenomonas ruminantium (S. rum), an enzyme that differs at key amino acid positions from canonical allosteric LDHs. The wild type (Wt) of this enzyme recognises pyuvate as all LDHs. However, introducing a single point mutation in the active site loop (I85R) allows S. Rum LDH to recognize the oxaloacetate substrate as a typical malate dehydrogenase (MalDH), whilst maintaining homotropic activation as an LDH. We report the tertiary structure of the Wt and I85RLDH mutant. The Wt S. rum enzyme structure binds NADH and malonate, whilst also resembling the typical compact R-active state of canonical LDHs. The structure of the mutant with I85R was solved in the Apo State (without ligand), and shows no large conformational reorganization such as that observed with canonical allosteric LDHs in Apo state. This is due to a local structural feature typical of S. rum LDH that prevents large-scale conformational reorganization. The S. rum LDH was also studied using Molecular Dynamics simulations, probing specific local deformations of the active site that allow the S. rum LDH to sample the T-inactive state. We propose that, with respect to the LDH/MalDH superfamily, the S. rum enzyme possesses a specificstructural and dynamical way to ensure homotropic activation.

Estimation of model accuracy in CASP15 using the ModFOLDdock server.

In CASP15, there was a greater emphasis on multimeric modeling than in previous experiments, with assembly structures nearly doubling in number (41 up from 22) since the previous round. CASP15 also included a new estimation of model accuracy (EMA) category in recognition of the importance of objective quality assessment (QA) for quaternary structure models. ModFOLDdock is a multimeric model QA server developed by the McGuffin group at the University of Reading, which brings together a range of single-model, clustering, and deep learning methods to form a consensus of approaches. For CASP15, three variants of ModFOLDdock were developed to optimize for the different facets of the quality estimation problem. The standard ModFOLDdock variant produced predicted scores optimized for positive linear correlations with the observed scores. The ModFOLDdockR variant produced predicted scores optimized for ranking, that is, the top-ranked models have the highest accuracy. In addition, the ModFOLDdockS variant used a quasi-single model approach to score each model on an individual basis. The scores from all three variants achieved strongly positive Pearson correlation coefficients with the CASP observed scores (oligo-lDDT) in excess of 0.70, which were maintained across both homomeric and heteromeric model populations. In addition, at least one of the ModFOLDdock variants was consistently ranked in the top two methods across all three EMA categories. Specifically, for overall global fold prediction accuracy, ModFOLDdock placed second and ModFOLDdockR placed third; for overall interface quality prediction accuracy, ModFOLDdockR, ModFOLDdock, and ModFOLDdockS were placed above all other predictor methods, and ModFOLDdockR and ModFOLDdockS were placed second and third respectively for individual residue confidence scores. The ModFOLDdock server is available at: https://www.reading.ac.uk/bioinf/ModFOLDdock/. ModFOLDdock is also available as part of the MultiFOLD docker package: https://hub.docker.com/r/mcguffin/multifold.

Combining pairwise structural similarity and deep learning interface contact prediction to estimate protein complex model accuracy in CASP15.

Estimating the accuracy of quaternary structural models of protein complexes and assemblies (EMA) is important for predicting quaternary structures and applying them to studying protein function and interaction. The pairwise similarity between structural models is proven useful for estimating the quality of protein tertiary structural models, but it has been rarely applied to predicting the quality of quaternary structural models. Moreover, the pairwise similarity approach often fails when many structural models are of low quality and similar to each other. To address the gap, we developed a hybrid method (MULTICOM_qa) combining a pairwise similarity score (PSS) and an interface contact probability score (ICPS) based on the deep learning inter-chain contact prediction for estimating protein complex model accuracy. It blindly participated in the 15th Critical Assessment of Techniques for Protein Structure Prediction (CASP15) in 2022 and performed very well in estimating the global structure accuracy of assembly models. The average per-target correlation coefficient between the model quality scores predicted by MULTICOM_qa and the true quality scores of the models of CASP15 assembly targets is 0.66. The average per-target ranking loss in using the predicted quality scores to rank the models is 0.14. It was able to select good models for most targets. Moreover, several key factors (i.e., target difficulty, model sampling difficulty, skewness of model quality, and similarity between good/bad models) for EMA are identified and analyzed. The results demonstrate that combining the multi-model method (PSS) with the complementary single-model method (ICPS) is a promising approach to EMA.

The impact of AI-based modeling on the accuracy of protein assembly prediction: Insights from CASP15.

In CASP15, 87 predictors submitted around 11 000 models on 41 assembly targets. The community demonstrated exceptional performance in overall fold and interface contact predictions, achieving an impressive success rate of 90% (compared to 31% in CASP14). This remarkable accomplishment is largely due to the incorporation of DeepMind's AF2-Multimer approach into custom-built prediction pipelines. To evaluate the added value of participating methods, we compared the community models to the baseline AF2-Multimer predictor. In over 1/3 of cases, the community models were superior to the baseline predictor. The main reasons for this improved performance were the use of custom-built multiple sequence alignments, optimized AF2-Multimer sampling, and the manual assembly of AF2-Multimer-built subcomplexes. The best three groups, in order, are Zheng, Venclovas, and Wallner. Zheng and Venclovas reached a 73.2% success rate over all (41) cases, while Wallner attained 69.4% success rate over 36 cases. Nonetheless, challenges remain in predicting structures with weak evolutionary signals, such as nanobody-antigen, antibody-antigen, and viral complexes. Expectedly, modeling large complexes also remains challenging due to their high memory compute demands. In addition to the assembly category, we assessed the accuracy of modeling interdomain interfaces in the tertiary structure prediction targets. Models on seven targets featuring 17 unique interfaces were analyzed. Best predictors achieved a 76.5% success rate, with the UM-TBM group being the leader. In the interdomain category, we observed that the predictors faced challenges, as in the case of the assembly category, when the evolutionary signal for a given domain pair was weak or the structure was large. Overall, CASP15 witnessed unprecedented improvement in interface modeling, reflecting the AI revolution seen in CASP14.

Peptide-based LDH5 inhibitors enter cancer cells and impair proliferation.

Lactate dehydrogenase 5 (LDH5) is overexpressed in many cancers and is a potential target for anticancer therapy due to its role in aerobic glycolysis. Small-molecule drugs have been developed as competitive inhibitors to bind substrate/cofactor sites of LDH5, but none reached the clinic to date. Recently, we designed the first LDH5 non-competitive inhibitor, cGmC9, a peptide that inhibits protein-protein interactions required for LDH5 enzymatic activity. Peptides are gaining a large interest as anticancer agents to modulate intracellular protein-protein interactions not targetable by small molecules; however, delivery of these peptides to the cytosol, where LDH5 and other anticancer targets are located, remains a challenge for this class of therapeutics. In this study, we focused on the cellular internalisation of cGmC9 to achieve LDH5 inhibition in the cytosol. We designed cGmC9 analogues and compared them for LDH5 inhibition, cellular uptake, toxicity, and antiproliferation against a panel of cancer cell lines. The lead analogue, [R/r]cGmC9, specifically impairs proliferation of cancer cell lines with high glycolytic profiles. Proteomics analysis showed expected metabolic changes in response to decreased glycolysis. This is the first report of a peptide-based LDH5 inhibitor able to modulate cancer metabolism and kill cancer cells that are glycolytic. The current study demonstrates the potential of using peptides as inhibitors of intracellular protein-protein interactions relevant for cancer pathways and shows that active peptides can be rationally designed to improve their cell permeation.

ATP-Binding Cassette Transporters: Snap-on Complexes?

ATP-binding cassette (ABC) transporters are one of the largest families of membrane proteins in prokaryotic organisms. Much is now understood about the structure of these transporters and many reviews have been written on that subject. In contrast, less has been written on the assembly of ABC transporter complexes and this will be a major focus of this book chapter. The complexes are formed from two cytoplasmic subunits that are highly conserved (in terms of their primary and three-dimensional structures) across the whole family. These ATP-binding subunits give rise to the name of the family. They must assemble with two transmembrane subunits that will typically form the permease component of the transporter. The transmembrane subunits have been found to be surprisingly diverse in structure when the whole family is examined, with seven distinct folds identified so far. Hence nucleotide-binding subunits appear to have been bolted on to a variety of transmembrane platforms during evolution, leading to a greater variety in function. Furthermore, many importers within the family utilise a further external substrate-binding component to trap scarce substrates and deliver them to the correct permease components. In this chapter, we will discuss whether assembly of the various ABC transporter subunits occurs with high fidelity within the crowded cellular environment and whether promiscuity in assembly of transmembrane and cytoplasmic components can occur. We also discuss the new AlphaFold protein structure prediction tool which predicts a new type of transmembrane domain fold within the ABC transporters that is associated with cation exporters of bacteria and plants.

Structural Characterization of Murine Phosphodiesterase 5 Isoforms and Involvement of Cysteine Residues in Supramolecular Assembly.

Phosphodiesterases (PDEs) are a superfamily of evolutionarily conserved cyclic nucleotide (cAMP/cGMP)-hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Within this family, the cGMP-dependent PDE5 is the major hydrolyzing enzyme in many mammalian tissues, where it regulates a number of cellular and tissular processes. Using Kluyveromyces lactis as a model organism, the murine PDE5A1, A2 and A3 isoforms were successfully expressed and studied, evidencing, for the first time, a distinct role of each isoform in the control, modulation and maintenance of the cellular redox metabolism. Moreover, we demonstrated that the short N-terminal peptide is responsible for the tetrameric assembly of MmPDE5A1 and for the mitochondrial localization of MmPDE5A2. We also analyzed MmPDE5A1, A2 and A3 using small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), structural mass spectrometry (MS) and polyacrylamide gel electrophoresis in their native conditions (native-PAGE) and in the presence of redox agents. These analyses pointed towards the role of a few specific cysteines in the isoforms' oligomeric assembly and the loss of enzymatic activity when modified.

Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme.

Mammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the monomeric form of the enzyme. Molecular dynamics simulations showed different geometries of the active site in the non-mutated trimeric and monomeric PNP forms, which suggested that the active site in the isolated monomer could be non-functional. To confirm this hypothesis, six amino acids located at the interface of the subunits were selected and mutated to alanines to disrupt the trimer and obtain a monomer (6Ala PNP). The effects of these mutations on the enzyme structure, stability, conformational dynamics, and activity were examined. The solution experiments confirmed that the 6Ala PNP mutant occurs mainly as a monomer, with a secondary structure almost identical to the wild type, WT PNP, and importantly, it shows no enzymatic activity. Simulations confirmed that, although the secondary structure of the 6Ala monomer is similar to the WT PNP, the positions of the amino acids building the 6Ala PNP active site significantly differ. These data suggest that a trimeric structure is necessary to stabilize the geometry of the active site of this enzyme.

The PAQosome, an R2TP-Based Chaperone for Quaternary Structure Formation.

The Rvb1-Rvb2-Tah1-Pih1/prefoldin-like (R2TP/PFDL) complex is a unique chaperone that provides a platform for the assembly and maturation of many key multiprotein complexes in mammalian cells. Here, we propose to rename R2TP/PFDL as PAQosome (particle for arrangement of quaternary structure) to more accurately represent its unique function.

Distance-based reconstruction of protein quaternary structures from inter-chain contacts.

Predicting the quaternary structure of protein complex is an important problem. Inter-chain residue-residue contact prediction can provide useful information to guide the ab initio reconstruction of quaternary structures. However, few methods have been developed to build quaternary structures from predicted inter-chain contacts. Here, we develop the first method based on gradient descent optimization (GD) to build quaternary structures of protein dimers utilizing inter-chain contacts as distance restraints. We evaluate GD on several datasets of homodimers and heterodimers using true/predicted contacts and monomer structures as input. GD consistently performs better than both simulated annealing and Markov Chain Monte Carlo simulation. Starting from an arbitrarily quaternary structure randomly initialized from the tertiary structures of protein chains and using true inter-chain contacts as input, GD can reconstruct high-quality structural models for homodimers and heterodimers with average TM-score ranging from 0.92 to 0.99 and average interface root mean square distance from 0.72 Å to 1.64 Å. On a dataset of 115 homodimers, using predicted inter-chain contacts as restraints, the average TM-score of the structural models built by GD is 0.76. For 46% of the homodimers, high-quality structural models with TM-score ≥ 0.9 are reconstructed from predicted contacts. There is a strong correlation between the quality of the reconstructed models and the precision and recall of predicted contacts. Only a moderate precision or recall of inter-chain contact prediction is needed to build good structural models for most homodimers. Moreover, GD improves the quality of quaternary structures predicted by AlphaFold2 on a Critical Assessment of Techniques for Protein Structure Prediction-Critical Assessments of Predictions of Interactions dataset.

Two PKA RIα holoenzyme states define ATP as an isoform-specific orthosteric inhibitor that competes with the allosteric activator, cAMP.

Protein kinase A (PKA) holoenzyme, comprised of a cAMP-binding regulatory (R)-subunit dimer and 2 catalytic (C)-subunits, is the master switch for cAMP-mediated signaling. Of the 4 R-subunits (RIα, RIß, RIIα, RIIß), RIα is most essential for regulating PKA activity in cells. Our 2 RIα2C2 holoenzyme states, which show different conformations with and without ATP, reveal how ATP/Mg2+ functions as a negative orthosteric modulator. Biochemical studies demonstrate how the removal of ATP primes the holoenzyme for cAMP-mediated activation. The opposing competition between ATP/cAMP is unique to RIα. In RIIß, ATP serves as a substrate and facilitates cAMP-activation. The isoform-specific RI-holoenzyme dimer interface mediated by N3A-N3A' motifs defines multidomain cross-talk and an allosteric network that creates competing roles for ATP and cAMP. Comparisons to the RIIß holoenzyme demonstrate isoform-specific holoenzyme interfaces and highlights distinct allosteric mechanisms for activation in addition to the structural diversity of the isoforms.

Structure, Oligomerization and Activity Modulation in N-Ribohydrolases.

Enzymes catalyzing the hydrolysis of the N-glycosidic bond in nucleosides and other ribosides (N-ribohydrolases, NHs) with diverse substrate specificities are found in all kingdoms of life. While the overall NH fold is highly conserved, limited substitutions and insertions can account for differences in substrate selection, catalytic efficiency, and distinct structural features. The NH structural module is also employed in monomeric proteins devoid of enzymatic activity with different physiological roles. The homo-oligomeric quaternary structure of active NHs parallels the different catalytic strategies used by each isozyme, while providing a buttressing effect to maintain the active site geometry and allow the conformational changes required for catalysis. The unique features of the NH catalytic strategy and structure make these proteins attractive targets for diverse therapeutic goals in different diseases.