Here, we introduce the third release of Kalium database (http://kaliumdb.org/), a manually curated comprehensive depository that accumulates data on polypeptide ligands of potassium channels. The major goal of this amplitudinous update is to summarize findings for natural polypeptide ligands of K+ channels, as well as data for the artificial derivatives of these substances obtained over the decades of exploration. We manually analyzed more than 700 original manuscripts and systematized the information on mutagenesis, production of radio- and fluorescently labeled derivatives, and the molecular pharmacology of K+ channel ligands. In result, data on more than 1200 substances were processed and added enriching the database content fivefold. We also included the electrophysiological data obtained on the understudied and neglected K+ channels including the heteromeric and concatenated channels. We associated target channels in Kalium with corresponding entries in the official database of the International Union of Basic and Clinical Pharmacology (IUPHAR). Kalium was supplemented with an adaptive Statistics page, where users are able to obtain actual data output. Several other improvements were introduced, such as a color code to distinguish the range of ligand activity concentrations and advanced tools for filtration and sorting. Kalium is a fully open-access database, crosslinked to other databases of interest. It can be utilized as a convenient resource containing ample up-to-date information about polypeptide ligands of K+ channels. This article is protected by copyright. All rights reserved.

We have applied our advanced computational and experimental methodologies to investigate the complex structure and binding mechanism of a modified Wilms' Tumor 1 (mWT1) protein epitope to the understudied Asian-dominant allele HLA-A*24:02 (HLA-A24) in aqueous solution. We have applied our developed multicanonical molecular dynamics (McMD)-based dynamic docking method to analyze the binding pathway and mechanism, which we verified by comparing the highest probability structures from simulation with our experimentally solved X-ray crystal structure. Subsequent path sampling MD simulations elucidated the atomic details of the binding process and indicated that first an encounter complex is formed between the N-terminal's positive charge of the 9-residue mWT1 fragment peptide and a cluster of negative residues on the surface of HLA-A24, with the MHC molecule preferring a predominantly closed conformation. The peptide first binds to this closed MHC conformation, forming an encounter complex, after which the binding site opens due to increased entropy of the binding site, allowing the peptide to bind to form the native complex structure. Further sequence and structure analyses also suggest that although the peptide loading complex would help with stabilizing the MHC molecule, the binding depends in a large part on the intrinsic affinity between the MHC molecule and the antigen peptide. Finally, our computational tools and analyses can be of great benefit to study the binding mechanism of different MHC types to their antigens, where it could also be useful in the development of higher affinity variant peptides and for personalized medicine. This article is protected by copyright. All rights reserved.

Predicting the effects of mutations on protein function and stability is an outstanding challenge. Here, we assess the performance of a variant of RoseTTAFold jointly trained for sequence and structure recovery, RFjoint , for mutation effect prediction. Without any further training, we achieve comparable accuracy in predicting mutation effects for a diverse set of protein families using RFjoint to both another zero-shot model (MSA Transformer) and a model which requires specific training on a particular protein family for mutation effect prediction (DeepSequence). Thus, although the architecture of RFjoint was developed to address the protein design problem of scaffolding functional motifs, RFjoint acquired an understanding of the mutational landscapes of proteins during model training that is equivalent to that of recently developed large protein language models. The ability to simultaneously reason over protein structure and sequence could enable even more precise mutation effect predictions following supervised training on the task. These results suggest that RFjoint has a quite broad understanding of protein sequence-structure landscapes, and can be viewed as a joint model for protein sequence and structure which could be broadly useful for protein modeling. This article is protected by copyright. All rights reserved.

In 2023, the Antimicrobial Peptide Database (currently available at https://aps.unmc.edu) is 20 years old. The timeline for the APD expansion in peptide entries, classification methods, search functions, post-translational modifications, binding targets, and mechanisms of action of antimicrobial peptides (AMPs) has been summarized in our previous Protein Science paper. This article highlights new database additions and findings. To facilitate antimicrobial development to combat drug-resistant pathogens, the APD has been re-annotating the data for antibacterial activity (active, inactive, and uncertain), toxicity (hemolytic and non-hemolytic AMPs), and salt tolerance (salt sensitive and insensitive). Comparison of the respective desired and undesired AMP groups produces new knowledge for peptide design. Our unification of AMPs from the six life kingdoms into "natural AMPs" enabled the first comparison with globular or transmembrane proteins. Due to the dominance of amphipathic helical and disulfide-linked peptides, cysteine, glycine, and lysine in natural AMPs are much more abundant than those in globular proteins. To include peptides predicted by machine learning, a new "predicted" group has been created. Remarkably, the averaged amino acid composition of predicted peptides is located between the lower bound of natural AMPs and the upper bound of synthetic peptides. Synthetic peptides in the current APD, with the highest cationic and hydrophobic amino acid percentages, are mostly designed with varying degrees of optimization. Hence, natural AMPs accumulated in the APD over 20 years have laid the foundation for machine learning prediction. We discuss future directions for peptide discovery. It is anticipated that the APD will continue to play a role in research and education. This article is protected by copyright. All rights reserved.

Malate (2-hydroxysuccinic acid) and tartrate (2,3-dihydroxysuccinic acid) are chiral substrates; the former existing in two enantiomeric forms (R and S) while the latter exists as three stereoisomers (R,R; S,S; and R,S). Dehydration by stereospecific hydrogen abstraction and anti-elimination of the hydroxyl group yield the achiral products fumarate and oxaloacetate, respectively. Class-I fumarate hydratase (FH) and L-tartrate dehydratase (L-TTD) are two highly conserved enzymes belonging to the iron-sulfur cluster hydrolyase family of enzymes that catalyze reactions on specific stereoisomers of malate and tartrate. FH from Methanocaldococcus jannaschii accepts only S-malate and S,S-tartrate as substrates while the structurally similar L-TTD from Escherichia coli accepts only R-malate and R,R-tartrate as substrates. Phylogenetic analysis reveals a common evolutionary origin of L-TTDs and two-subunit archaeal FHs suggesting a divergence during evolution that may have led to the switch in substrate stereospecificity preference. Due to the high conservation of their sequences, a molecular basis for switch in stereospecificity is not evident from analysis of crystal structures of FH and predicted structure of L-TTD. The switch in enantiomer preference may be rationalised by invoking conformational plasticity of the amino acids interacting with the substrate, together with substrate reorientation and conformer selection about the C2-C3 bond of the dicarboxylic acid substrates. Although classical models of enzyme-substrate binding are insufficient to explain such a phenomenon, the enantiomer superposition model suggests that a minor reorientation in the active site residues could lead to the switch in substrate stereospecificity. This article is protected by copyright. All rights reserved.

Small-molecule responsive protein switches are powerful tools for controlling cellular processes. These switches are designed to respond rapidly and specifically to their inducer. They have been used in numerous applications, including the regulation of gene expression, post-translational protein modification, and signal transduction. Typically, small-molecule responsive protein switches consist of two proteins that interact with each other in the presence or absence of a small-molecule. Recent advances in computational protein design already contributed to the development of protein switches with an expanded range of small-molecule inducers and increasingly sophisticated switch mechanisms. Further progress in the engineering of small-molecule responsive switches is fueled by cutting-edge computational design approaches, which will enable more complex and precise control over cellular processes and advance synthetic biology applications in biotechnology and medicine. Here, we discuss recent milestones and how technological advances are impacting the development of chemical switches. This article is protected by copyright. All rights reserved.

Recent studies have suggested that neuropilin-1 (NRP1) may serve as a potential receptor in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. However, the biophysical characteristics of interactions between NRP1 and SARS-CoV-2 remain unclear. In this study, we examined the interactions between NRP1 and various SARS-CoV-2 spike (S) fragments, including the receptor-binding domain (RBD) and the S protein trimer in a soluble form or expressed on pseudovirions, using atomic force microscopy (AFM) and structural modeling. Our measurements showed that NRP1 interacts with the RBD and trimer at a higher binding frequency compared to ACE2. This NRP1-RBD interaction has also been predicted and simulated via AlphaFold2 and molecular dynamic simulations, and the results indicate that their binding patterns are very similar to RBD-ACE2 interactions. Additionally, under similar loading rates, the most probable unbinding forces between NRP1 and S trimer (both soluble form and on pseudovirions) are larger than the forces between NRP1 and RBD and between trimer and ACE2. Further analysis indicates that NRP1 has a stronger binding affinity to the SARS-CoV-2 S trimer with a dissociation rate of 0.87 s-1 , four times lower than the dissociation rate of 3.65 s-1 between NRP1 and RBD. Moreover, additional experiments show that RBD-neutralizing antibodies can significantly reduce the binding frequency for both ACE2 and NRP1. Together, the study suggests that NRP1 can be an alternative receptor for SARS-CoV-2 attachment to human cells, and the neutralizing antibodies targeting SARS-CoV-2 RBD can reduce the binding between SARS-CoV-2 and NRP1. This article is protected by copyright. All rights reserved.

Efficient identification of epitopes is crucial for drug discovery and design as it enables the selection of optimal epitopes, expansion of lead antibody diversity, and verification of binding interface. Although high resolution low throughput methods like X-ray crystallography can determine epitopes or protein-protein interactions accurately, they are time-consuming and can only be applied to a limited number of complexes. To overcome these limitations, we have developed a rapid computational method that incorporates N-linked glycans to mask epitopes or protein interaction surfaces, thereby providing a mapping of these regions. Using human coagulation factor IXa (fIXa) as a model system, we computationally screened 158 positions and expressed 98 variants to test experimentally for epitope mapping. We were able to delineate epitopes rapidly and reliably through the insertion of N-linked glycans that efficiently disrupted binding in a site-selective manner. To validate the efficacy of our method, we conducted ELISA experiments and high-throughput yeast surface display assays. Furthermore, X-ray crystallography was employed to verify the results, thereby recapitulating through the method of N-linked glycans a coarse-grained mapping of the epitope. This article is protected by copyright. All rights reserved.

Many proteins that self-assemble into amyloid and amyloid-like fibres can adopt diverse polymorphic forms. These forms have been observed both in vitro and in vivo and can arise through variations in the steric-zipper interactions between ꞵ-sheets, variations in the arrangements between protofilaments, and differences in the number of protofilaments that make up a given fibre class. Different polymorphs arising from the same precursor molecule not only exhibit different levels of toxicity, but importantly can contribute to different disease conditions. However, the factors which contribute to formation of polymorphic forms of amyloid fibrils are not known. In this work, we show that in the presence of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine, a highly abundant lipid in the plasma membrane of neurons, the aggregation of α-synuclein is markedly accelerated and yields a diversity of polymorphic forms under identical experimental conditions. This morphological diversity includes thin and curly fibrils, helical ribbons, twisted ribbons, nanotubes, and flat sheets. Furthermore, the amyloid fibrils formed incorporate lipids into their structures, which corroborates the previous report of the presence of α-synuclein fibrils with high lipid content in Lewy bodies. Thus, the present study demonstrates that an interface, such as that provided by a lipid membrane, can not only modulate the kinetics of α-synuclein amyloid aggregation but also plays an important role in the formation of morphological variants by incorporating lipid molecules in the process of amyloid fibril formation. This article is protected by copyright. All rights reserved.

Carboxylate-bridged diiron proteins belong to a protein family involved in different physiological processes. These proteins share the conservative EXXH motif, which provides the carboxylate bridge and is critical for metal binding. Here, we choose de novo-designed due ferri protein DFsc, a four-helical protein with two EXXH motifs as a model protein, to study the stability of the carboxylate-bridged di-metal binding site. The mechanical and kinetic properties of the di-Zn site in DFsc were obtained by AFM-based single-molecule force spectroscopy. Zn-DFsc showed a considerable rupture force of ~200 pN, while the apo-protein is mechanically labile. In addition, multiple rupture pathways were observed with different probabilities, indicating the importance of the EXXH-based carboxylate-bridged metal site. These results demonstrate carboxylate-bridged di-metal site is mechanically stable and improve our understanding of this important type of metalloprotein. This article is protected by copyright. All rights reserved.

The ConSurf web-sever for the analysis of proteins, RNA, and DNA provides a quick and accurate estimate of the per-site evolutionary rate among homologues. The analysis reveals functionally important regions, such as catalytic and ligand-binding sites, which often evolve slowly. Since the last report in 2016, ConSurf has been improved in multiple ways. It now has a user-friendly interface that makes it easier to perform the analysis and to visualize the results. Evolutionary rates are calculated based on a set of homologous sequences, collected using hidden Markov model-based search tools, recently embedded in the pipeline. Using these, and following the removal of redundancy, ConSurf assembles a representative set of effective homologues for protein and nucleic acid queries to enable informative analysis of the evolutionary patterns. The analysis is particularly insightful when the evolutionary rates are mapped on the macromolecule structure. In this respect, the availability of AlphaFold model structures of essentially all UniProt proteins makes ConSurf particularly relevant to the research community. The UniProt ID of a query protein with an available AlphaFold model can now be used to start a calculation. Another important improvement is the Python re-implementation of the entire computational pipeline, making it easier to maintain. This Python pipeline is now available for download as a standalone version. We demonstrate some of ConSurf's key capabilities by the analysis of caveolin-1, the main protein of membrane invaginations called caveolae. The ConSurf web-site is available at: https://consurf.tau.ac.il The standalone pipeline is available at: https://consurf.tau.ac.il/STANDALONE/stand_alone_consurf-1.00.rar This article is protected by copyright. All rights reserved.

The fungicide nucleoside blasticidin S features a β-arginine, a moiety seldom revealed in the structure of natural products. BlsG, a radical SAM arginine-2,3-aminomutase from the blasticidin S biosynthetic pathway, displayed promiscuous activity to three basic amino acids. Here in this study, we demonstrated that BlsG showed high preference toward its natural substrate arginine. The combined structural modeling, steady-state kinetics, and mutational analyses lead to the detailed understanding of the substrate recognition of BlsG. A single mutation of T340D changed the substrate preference of BlsG leading to a little more preference to lysine than arginine. On the basis of our understanding of the substrate selection of BlsG and bioinformatic analysis, we propose that the D…D motif locationally corresponding to D293 and D330 of KAM is characteristic of lysine 2,3-aminomutase while the corresponding D…T motif is characteristic of arginine 2,3-aminomutase. The study may provide a simple way to discern the arginine 2,3-aminomutase and thus lead to the discovery of new natural compounds with β-arginine moiety. This article is protected by copyright. All rights reserved.

Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple-drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/β-fold with multiple secondary structure elements omitted or shortened compared to protein structures of similar proteins. The functional characterisation of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity towards thermophilic and mesophilic bacteria strains containing Orn-type or DAP-type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterised with a temperature optimum at 85 °C. The extraordinary high melting temperature Tm 102.6 °C confirms fold stability up to approximately 100 °C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology. This article is protected by copyright. All rights reserved.

In addition to their membrane-bound chlorophyll a/c light-harvesting antenna, the cryptophyte algae have evolved a unique phycobiliprotein antenna system located in the thylakoid lumen. The basic unit of this antenna consists of two copies of an αβ protomer where the α and β subunits scaffold different combinations of a limited number of linear tetrapyrrole chromophores. While the β subunit is highly conserved, encoded by a single plastid gene, the nuclear-encoded α subunits have evolved diversified multigene families. It is still unclear how this sequence diversity results in the spectral diversity of the mature proteins. By careful examination of three newly-determined crystal structures in comparison with three previously obtained, we show how the α subunit amino acid sequences control chromophore conformations and hence spectral properties even when the chromophores are identical. Previously we have shown that α subunits control the quaternary structure of the mature αβ.αβ complex (either open or closed), however, each species appeared to only harbor a single quaternary form. Here we show that species of the Hemiselmis genus contain expressed α subunit genes that encode both distinct quaternary structures. Finally, we have discovered a common single-copy gene (expressed into protein) consisting of tandem copies of a small α subunit that could potentially scaffold pairs of light harvesting units. Together, our results show how the diversity of the multigene α subunit family produces a range of mature cryptophyte antenna proteins with differing spectral properties, and the potential for minor forms that could contribute to acclimation to varying light regimes. This article is protected by copyright. All rights reserved.

In photosynthesis, pigment - protein complexes achieve outstanding photoinduced charge separation efficiencies through a set of strategies in which excited states delocalisation over multiple pigments ('excitons') and charge-transfer states play key roles. These concepts, and their implementation in bioinspired artificial systems, are attracting increasing attention due to the vast potential that could be tapped by realising efficient photochemical reactions. In particular, de novo designed proteins provide a diverse structural toolbox that can be used to manipulate the geometric and electronic properties of bound chromophore molecules. However, achieving excitonic and charge-transfer states requires closely spaced chromophores, a non-trivial aspect since a strong binding with the protein matrix needs to be maintained. Here, we show how a general-purpose artificial protein can be optimised via molecular dynamics simulations to improve its binding capacity of a chlorophyll derivative, achieving complexes in which chromophores form two closely spaced and strongly interacting dimers. Based on spectroscopy results and computational modelling, we demonstrate each dimer is excitonically coupled, and propose they display signatures of charge-transfer state mixing. This work could open new avenues for the rational design of chromophore - protein complexes with advanced functionalities. This article is protected by copyright. All rights reserved.

Tau, as typical of intrinsically disordered proteins (IDPs), binds to multiple targets including microtubules and acidic membranes. The latter two surfaces are both highly negatively charged, raising the prospect of mimicry in their binding by tau. The tau-microtubule complex was recently determined by cryo-EM. Here we used molecular dynamics simulations to characterize the dynamic binding of tau K19 to an acidic membrane. This IDP can be divided into three repeats, each containing an amphipathic helix. The three amphipathic helices, along with flanking residues, tether the protein to the membrane interface. The separation between and membrane positioning of the amphipathic helices in the simulations are validated by published EPR data. The membrane contact probabilities of individual residues in tau show both similarities to and distinctions from native contacts with microtubules. In particular, a Lys that is conserved among the repeats forms similar interactions with membranes and with microtubules, as does a conserved Val. This partial mimicry facilitates both the membrane anchoring of microtubules by tau and the transfer of tau from membranes to microtubules. This article is protected by copyright. All rights reserved.

Unlike globular proteins, mutational effects on the function of Intrinsically Disordered Proteins (IDPs) are not well-studied. Deep Mutational Scanning of a yeast surface displayed mutant library yields insights into sequence-function relationships in the CcdA IDP. The approach enables facile prediction of interface residues and local structural signatures of the bound conformation. In contrast to previous titration-based approaches which use a number of ligand concentrations, we show that use of a single rationally chosen ligand concentration can provide quantitative estimates of relative binding constants for large numbers of protein variants. This is because the extended interface of IDP ensures that energetic effects of point mutations are spread over a much smaller range than for globular proteins. Our data also provides insights into the much-debated role of helicity and disorder in partner binding of IDPs. Based on this exhaustive mutational sensitivity dataset, a rudimentary model was developed in an attempt to predict mutational effects on binding affinity of IDPs that form alpha-helical structures upon binding. This article is protected by copyright. All rights reserved.

Immune checkpoint inhibitors that bind to the cell surface receptor PD-L1 are effective anti-cancer agents but suffer from immune related adverse events as PD-L1 is expressed on both healthy and cancer cells. To mitigate toxicity, researchers are testing prodrugs that have low affinity for checkpoint targets until activated with proteases enriched in the tumor microenvironment. Here, we engineer a prodrug form of a PD-L1 inhibitor. The inhibitor is a soluble PD-1 mimetic that was previously engineered to have high affinity for PD-L1. In the basal state, the binding surface of the PD-1 mimetic is masked by fusing it to a soluble variant of its natural ligand, PD-L1. Proteolytic cleavage of the linker that connects the mask to the inhibitor activates the molecule. To optimize the mask so that it effectively blocks binding to PD-L1 but releases upon cleavage, we tested a set of mutants with varied affinity for the inhibitor. The top-performing mask reduces affinity of the prodrug for PD-L1 120-fold, and binding is nearly fully recovered upon cleavage. In a cell-based assay measuring inhibition of the PD-1:PD-L1 interaction on the surface of cells, the IC50s of the masked inhibitors were up to 40-fold higher than their protease-treated counterparts. The changes in activity we observe upon protease treatment are comparable to systems currently tested in the clinic and provide evidence that natural binding partners are an excellent starting point for creating a prodrug. This article is protected by copyright. All rights reserved.

An integrative approach to visualization is used to create a visual snapshot of the structural biology of the polar microdomain of Caulobacter crescentus. The visualization is based on the current state of molecular and cellular knowledge of the microdomain and its cellular context. The collaborative process of researching and executing the visualization has identified aspects that are well determined and areas that require further study. The visualization is useful for dissemination, education, and outreach, and the study lays the groundwork for future 3D modeling and simulation of this well-studied example of a cellular condensate. This article is protected by copyright. All rights reserved.

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase and its cofactor, Cdh1, regulate the expression of several cell-cycle proteins and their functions during mitosis. Levels of the protein cell division cycle-associated protein 3 (CDCA3), which is functionally required for mitotic entry, are regulated by APC/CCdh1 . CDCA3 is an intrinsically disordered protein and contains both C-terminal KEN box and D-box recognition motifs, enabling binding to Cdh1. Our previous findings demonstrate that CDCA3 has a phosphorylation-dependent non-canonical ABBA-like motif within the linker region bridging these two recognition motifs and is required for efficient binding to Cdh1. Here, we sought to identify and further characterize additional residues that participate within this ABBA-like motif using detailed in vitro experiments and in silico modelling studies. We identified the role of H-bonds, hydrophobic and ionic interactions across the CDCA3 ABBA-like motif in the linker region between KEN and D-box motifs. This linker region adopts a well-defined structure when bound to Cdh1 in the presence of phosphorylation. Upon alanine mutation, the structure of this region is lost, leading to higher flexibility, and alteration in affinities due to binding to alternate sites on Cdh1. Our findings identify roles for the anchoring residues in the non-canonical ABBA-like motif to promote binding to the APC/CCdh1 and regulation of CDCA3 protein levels. This article is protected by copyright. All rights reserved.