Search

Abstract Human proteins are crucial players in both health and disease. Understanding their molecular landscape is a central topic in biological research. Here, we present an extensive dataset of predicted protein structures for 42,042 distinct human proteins, including splicing variants, derived from the UniProt reference proteome UP000005640. To ensure high quality and comparability, the dataset was generated by combining state-of-the-art modeling-tools AlphaFold 2, OpenFold, and ESMFold, provided within NVIDIA’s BioNeMo platform, as well as homology modeling using Innophore’s CavitomiX platform. Our dataset is offered in both unedited and edited formats for diverse research requirements. The unedited version contains structures as generated by the different prediction methods, whereas the edited version contains refinements, including a dataset of structures without low prediction-confidence regions and structures in complex with predicted ligands based on homologs in the PDB. We are confident that this dataset represents the most comprehensive collection of human protein structures available today, facilitating diverse applications such as structure-based drug design and the prediction of protein function and interactions.

Abstract According to the World Health Organization, Chagas disease (CD) is the most prevalent poverty-promoting neglected tropical disease. Alarmingly, climate change is accelerating the geographical spreading of CD causative parasite, Trypanosoma cruzi, which additionally increases infection rates. Still, CD treatment remains challenging due to a lack of safe and efficient drugs. In this work, we analyze the viability of T. cruzi Akt-like kinase (TcAkt) as drug target against CD including primary structural and functional information about a parasitic Akt protein. Nuclear Magnetic Resonance derived information in combination with Molecular Dynamics simulations offer detailed insights into structural properties of the pleckstrin homology (PH) domain of TcAkt and its binding to phosphatidylinositol phosphate ligands (PIP). Experimental data combined with Alpha Fold proposes a model for the mechanism of action of TcAkt involving a PIP-induced disruption of the intramolecular interface between the kinase and the PH domain resulting in an open conformation enabling TcAkt kinase activity. Further docking experiments reveal that TcAkt is recognized by human inhibitors PIT-1 and capivasertib, and TcAkt inhibition by UBMC-4 and UBMC-6 is achieved via binding to TcAkt kinase domain. Our in-depth structural analysis of TcAkt reveals potential sites for drug development against CD, located at activity essential regions.

Advancing climate change increases the risk of future infectious disease outbreaks, particularly of zoonotic diseases, by affecting the abundance and spread of viral vectors. Concerningly, there are currently no approved drugs for some relevant diseases, such as the arboviral diseases chikungunya, dengue or zika. The development of novel inhibitors takes 10–15 years to reach the market and faces critical challenges in preclinical and clinical trials, with approximately 30% of trials failing due to side effects. As an early response to emerging infectious diseases, CavitOmiX allows for a rapid computational screening of databases containing 3D point-clouds representing binding sites of approved drugs to identify candidates for off-label use. This process, known as drug repurposing, reduces the time and cost of regulatory approval. Here, we present potential approved drug candidates for off-label use, targeting the ADP-ribose binding site of Alphavirus chikungunya non-structural protein 3. Additionally, we demonstrate a novel in silico drug design approach, considering potential side effects at the earliest stages of drug development. We use a genetic algorithm to iteratively refine potential inhibitors for (i) reduced off-target activity and (ii) improved binding to different viral variants or across related viral species, to provide broad-spectrum and safe antivirals for the future.

ABSTRACT The monkeypox virus (MPX) belongs to the Orthopoxvirus genus of the Poxviridae family, is endemic in parts of Africa and causes a disease in humans similar to smallpox. The most recent outbreak of MPX is already affecting 110 countries, with 86,956 confirmed cases since May 2022 and has consequently become a focus of interest. In particular, a molecular understanding of the virus is essential to study infection processes and pathogen-host interactions, predict tropism changes, or guide drug development and drug discovery as well as vaccine development or vaccine adaptation at a very early stage. Herein, we present a study of the structural proteome of the currently circulating MPX: Our consensus analysis of 3,713 genome sequences sampled within a year after the outbreak revealed 10,580 characteristic candidate open reading frames (ORFs). A search in the non-redundant protein database reduced the number of suspected ORFs to 1,079, of which 210 are representative proteins in typical MPX reference genomes. This should serve as a collection of putative proteins within the currently spreading MPX, a compound of information that could support timely drug discovery, mutational analyses, and vaccine development. We, herein, present the so far most comprehensive structural proteome by providing atomistic 3D models of 210 proteins, generated with three state-of-the-art structure prediction methods, including a mutational analysis of the proteome, with a particular focus on the drug-binding sites of tecovirimat and brincidofovir. IMPORTANCE The 2022 outbreak of the monkeypox virus already involves, by April 2023, 110 countries with 86,956 confirmed cases and 119 deaths. Understanding an emerging disease on a molecular level is essential to study infection processes and eventually guide drug discovery at an early stage. To support this, we provide the so far most comprehensive structural proteome of the monkeypox virus, which includes 210 structural models, each computed with three state-of-the-art structure prediction methods. Instead of building on a single-genome sequence, we generated our models from a consensus of 3,713 high-quality genome sequences sampled from patients within 1 year of the outbreak. Therefore, we present an average structural proteome of the currently isolated viruses, including mutational analyses with a special focus on drug-binding sites. Continuing dynamic mutation monitoring within the structural proteome presented here is essential to timely predict possible physiological changes in the evolving virus.

Abstract Treatment of COVID-19 with a soluble version of ACE2 that binds to SARS-CoV-2 virions before they enter host cells is a promising approach, however it needs to be optimized and adapted to emerging viral variants. The computational workflow presented here consists of molecular dynamics simulations for spike RBD-hACE2 binding affinity assessments of multiple spike RBD/hACE2 variants and a novel convolutional neural network architecture working on pairs of voxelized force-fields for efficient search-space reduction. We identified hACE2-Fc K31W and multi-mutation variants as high-affinity candidates, which we validated in vitro with virus neutralization assays. We evaluated binding affinities of these ACE2 variants with the RBDs of Omicron BA.3, Omicron BA.4/BA.5, and Omicron BA.2.75 in silico. In addition, candidates produced in Nicotiana benthamiana, an expression organism for potential large-scale production, showed a 4.6-fold reduction in half-maximal inhibitory concentration (IC50) compared with the same variant produced in CHO cells and an almost six-fold IC50 reduction compared with wild-type hACE2-Fc.

Abstract To date, more than 263 million people have been infected with SARS-CoV-2 during the COVID-19 pandemic. In many countries, the global spread occurred in multiple pandemic waves characterized by the emergence of new SARS-CoV-2 variants. Here we report a sequence and structural-bioinformatics analysis to estimate the effects of amino acid substitutions on the affinity of the SARS-CoV-2 spike receptor binding domain (RBD) to the human receptor hACE2. This is done through qualitative electrostatics and hydrophobicity analysis as well as molecular dynamics simulations used to develop a high-precision empirical scoring function (ESF) closely related to the linear interaction energy method and calibrated on a large set of experimental binding energies. For the latest variant of concern (VOC), B.1.1.529 Omicron, our Halo difference point cloud studies reveal the largest impact on the RBD binding interface compared to all other VOC. Moreover, according to our ESF model, Omicron achieves a much higher ACE2 binding affinity than the wild type and, in particular, the highest among all VOCs except Alpha and thus requires special attention and monitoring.

IntroductionThe current coronavirus pandemic is being combated worldwide by nontherapeutic measures and massive vaccination programs. Nevertheless, therapeutic options such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main-protease (Mpro) inhibitors are essential due to the ongoing evolution toward escape from natural or induced immunity. While antiviral strategies are vulnerable to the effects of viral mutation, the relatively conserved Mpro makes an attractive drug target: Nirmatrelvir, an antiviral targeting its active site, has been authorized for conditional or emergency use in several countries since December 2021, and a number of other inhibitors are under clinical evaluation. We analyzed recent SARS-CoV-2 genomic data, since early detection of potential resistances supports a timely counteraction in drug development and deployment, and discovered accelerated mutational dynamics of Mpro since early December 2021.MethodsWe performed a comparative analysis of 10.5 million SARS-CoV-2 genome sequences available by June 2022 at GISAID to the NCBI reference genome sequence NC_045512.2. Amino-acid exchanges within high-quality regions in 69,878 unique Mpro sequences were identified and time- and in-depth sequence analyses including a structural representation of mutational dynamics were performed using in-house software.ResultsThe analysis showed a significant recent event of mutational dynamics in Mpro. We report a remarkable increase in mutational variability in an eight-residue long consecutive region (R188-G195) near the active site since December 2021.DiscussionThe increased mutational variability in close proximity to an antiviral-drug binding site as described herein may suggest the onset of the development of antiviral resistance. This emerging diversity urgently needs to be further monitored and considered in ongoing drug development and lead optimization.

The AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis that initiates cytoplasmic maturation of the large subunit. Here the authors report the structure of Drg1 in complex with its specific inhibitor diazaborine and provide insight into the mechanism of inhibition and specificity of this class of inhibitors.

Abstract Since the worldwide outbreak of the infectious disease COVID-19, several studies have been published to understand the structural mechanism of the novel coronavirus SARS-CoV-2. During the infection process, the SARS-CoV-2 spike (S) protein plays a crucial role in the receptor recognition and cell membrane fusion process by interacting with the human angiotensin-converting enzyme 2 (hACE2) receptor. However, new variants of these spike proteins emerge as the virus passes through the disease reservoir. This poses a major challenge for designing a potent antigen for an effective immune response against the spike protein. Through a normal mode analysis (NMA) we identified the highly flexible region in the receptor binding domain (RBD) of SARS-CoV-2, starting from residue 475 up to residue 485. Structurally, the position S477 shows the highest flexibility among them. At the same time, S477 is hitherto the most frequently exchanged amino acid residue in the RBDs of SARS-CoV-2 mutants. Therefore, using MD simulations, we have investigated the role of S477 and its two frequent mutations (S477G and S477N) at the RBD during the binding to hACE2. We found that the amino acid exchanges S477G and S477N strengthen the binding of the SARS-COV-2 spike with the hACE2 receptor.

Environmentally friendly functionalization and recycling processes for synthetic polymers have recently gained momentum, and enzymes play a central role in these procedures. However, natural enzymes must be engineered to accept synthetic polymers as substrates. To enhance the activity on synthetic polyesters, the canonical amino acid methionine in Thermoanaerobacter thermohydrosulfuricus lipase (TTL) was exchanged by the residue-specific incorporation method for the more hydrophobic non-canonical norleucine (Nle). Strutural modelling of TTL revealed that residues Met-114 and Met-142 are in close vicinity of the active site and their replacement by the norleucine could modulate the catalytic activity of the enzyme. Indeed, hydrolysis of the polyethylene terephthalate model substrate by the Nle variant resulted in significantly higher amounts of release products than the Met variant. A similar trend was observed for an ionic phthalic polyester containing a short alkyl diol (C5). Interestingly, a 50% increased activity was found for TTL [Nle] towards ionic phthalic polyesters containing different ether diols compared to the parent enzyme TTL [Met]. These findings clearly demonstrate the high potential of non-canonical amino acids for enzyme engineering.

Carboxylate reductases (CARs, E.C. 1.2.1.30) generate aldehydes from their corresponding carboxylic acid with high selectivity. Little is known about the structure of CARs and their catalytically important amino acid residues. The identification of key residues for carboxylate reduction provides a starting point to gain deeper understanding of enzymatic carboxylate reduction. A multiple sequence alignment of CARs with confirmed activity recently identified in our lab and from the literature revealed a fingerprint of conserved amino acids. We studied the function of conserved residues by multiple sequence alignments and mutational replacements of these residues. In this study, single-site alanine variants of Neurospora crassa CAR were investigated to determine the contribution of conserved residues to the function, expressability or stability of the enzyme. The effect of amino acid replacements was investigated by analyzing enzymatic activity of the variants in vivo and in vitro. Supported by molecular modeling, we interpreted that five of these residues are essential for catalytic activity, or substrate and co-substrate binding. We identified amino acid residues having significant impact on CAR activity. Replacement of His 237, Glu 433, Ser 595, Tyr 844, and Lys 848 by Ala abolish CAR activity, indicating their key role in acid reduction. These results may assist in the functional annotation of CAR coding genes in genomic databases. While some other conserved residues decreased activity or had no significant impact, four residues increased the specific activity of NcCAR variants when replaced by alanine. Finally, we showed that NcCAR wild-type and mutants efficiently reduce aliphatic acids.

Friedel-Crafts alkylation of aromatic systems is a classic reaction in organic chemistry, for which regiospecific mono-alkylation, however, is generally difficult to achieve. In nature, methyltransferases catalyze the addition of methyl groups to a wide range of biomolecules thereby modulating the physico-chemical properties of these compounds. Specifically, S-adenosyl-L-methionine dependent C-methyltransferases possess a high potential to serve as biocatalysts in environmentally benign organic syntheses. Here, we report on the high resolution crystal structure of CouO, a C-methyltransferase from Streptomyces rishiriensis involved in the biosynthesis of the antibiotic coumermycin A1. Through molecular docking calculations, site-directed mutagenesis and the comparison with homologous enzymes we identified His120 and Arg121 as key functional residues for the enzymatic activity of this group of C-methyltransferases. The elucidation of the atomic structure and the insight into the catalytic mechanism provide the basis for the (semi)-rational engineering of the enzyme in order to increase the substrate scope as well as to facilitate the acceptance of SAM-analogues as alternative cofactors.

Bacteroides thetaiotaomicron is a dominant member of the human intestinal microbiome. The genome of this anaerobe encodes more than 100 proteolytic enzymes, the majority of which have not been characterized. In the present study, we have produced and purified recombinant dipeptidyl peptidase III (DPP III) from B. thetaiotaomicron for the purposes of biochemical and structural investigations. DPP III is a cytosolic zinc-metallopeptidase of the M49 family, involved in protein metabolism. The biochemical results for B. thetaiotaomicron DPP III from our research showed both some similarities to, as well as certain differences from, previously characterised yeast and human DPP III. The 3D-structure of B. thetaiotaomicron DPP III was determined by X-ray crystallography and revealed a two-domain protein. The ligand-free structure (refined to 2.4 Å) was in the open conformation, while in the presence of the hydroxamate inhibitor Tyr-Phe-NHOH, the closed form (refined to 3.3 Å) was observed. Compared to the closed form, the two domains of the open form are rotated away from each other by about 28 degrees. A comparison of the crystal structure of B. thetaiotaomicron DPP III with that of the human and yeast enzymes revealed a similar overall fold. However, a significant difference with functional implications was discovered in the upper domain, farther away from the catalytic centre. In addition, our data indicate that large protein flexibility might be conserved in the M49 family.

Porphyromonas gingivalis, an asaccharolytic Gram-negative oral anaerobe, is a major pathogen associated with adult periodontitis, a chronic infective disease that a significant percentage of the human population suffers from. It preferentially utilizes dipeptides as its carbon source, suggesting the importance of dipeptidyl peptidase (DPP) types of enzyme for its growth. Until now DPP IV, DPP5, 7 and 11 have been extensively investigated. Here, we report the characterization of DPP III using molecular biology, biochemical, biophysical and computational chemistry methods. In addition to the expected evolutionarily conserved regions of all DPP III family members, PgDPP III possesses a C-terminal extension containing an Armadillo (ARM) type fold similar to the AlkD family of bacterial DNA glycosylases, implicating it in alkylation repair functions. However, complementation assays in a DNA repair-deficient Escherichia coli strain indicated the absence of alkylation repair function for PgDPP III. Biochemical analyses of recombinant PgDPP III revealed activity similar to that of DPP III from Bacteroides thetaiotaomicron, and in the range between activities of human and yeast counterparts. However, the catalytic efficiency of the separately expressed DPP III domain is ~1000-fold weaker. The structure and dynamics of the ligand-free enzyme and its complex with two different diarginyl arylamide substrates was investigated using small angle X-ray scattering, homology modeling, MD simulations and hydrogen/deuterium exchange (HDX). The correlation between the experimental HDX and MD data improved with simulation time, suggesting that the DPP III domain adopts a semi-closed or closed form in solution, similar to that reported for human DPP III. The obtained results reveal an atypical DPP III with increased structural complexity: its superhelical C-terminal domain contributes to peptidase activity and influences DPP III interdomain dynamics. Overall, this research reveals multifunctionality of PgDPP III and opens direction for future research of DPP III family proteins.

Soluble water-forming NAD(P)H oxidases constitute a promising NAD(P)+ regeneration method as they only need oxygen as cosubstrate and produce water as sole byproduct. Moreover, the thermodynamic equilibrium of O2 reduction is a valuable driving force for mostly energetically unfavorable biocatalytic oxidations. Here, we present the generation of an NAD(P)H oxidase with high activity for both cofactors, NADH and NADPH. Starting from the strictly NADH specific water-forming Streptococcus mutans NADH oxidase 2 several rationally designed cofactor binding site mutants were created and kinetic values for NADH and NADPH conversion were determined. Double mutant 93R94H showed comparable high rates and low Km values for NADPH (kcat 20 s−1, Km 6 μM) and NADH (kcat 25 s−1, Km 9 μM) with retention of 70 % of wild type activity towards NADH. Moreover, by screening of a SeSaM library S. mutans NADH oxidase 2 variants showing predominantly NADPH activity were found, giving further insight into cofactor binding site architecture. Applicability for cofactor regeneration is shown for coupling with alcohol dehydrogenase from Sphyngobium yanoikuyae for 2-heptanone production.

Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.

Threonine aldolases catalyze the pyridoxal phosphate (PLP) dependent cleavage of threonine into glycine and acetaldehyde and play a major role in the degradation of this amino acid. In nature, L- as well as D-specific enzymes have been identified, but the exact physiological function of D-threonine aldolases (DTAs) is still largely unknown. Both types of enantio-complementary enzymes have a considerable potential in biocatalysis for the stereospecific synthesis of various β-hydroxy amino acids, which are valuable building blocks for the production of pharmaceuticals. While several structures of L-threonine aldolases (LTAs) have already been determined, no structure of a DTA is available to date. Here, we report on the determination of the crystal structure of the DTA from Alcaligenes xylosoxidans (AxDTA) at 1.5 Å resolution. Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity. Modeling of AxDTA substrate complexes provides a rationale for this metal dependence and indicates that binding of the β-hydroxy group of the substrate to the metal ion very likely activates this group and facilitates its deprotonation by His193. An equivalent involvement of a metal ion has been implicated in the mechanism of a serine dehydratase, which harbors a metal ion binding site in the vicinity of the PLP cofactor at the same position as in DTA. The structure of AxDTA is completely different to available structures of LTAs. The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.

Chiral amines are important building blocks for the synthesis of pharmaceutical products, fine chemicals, and agrochemicals. ω-Transaminases are able to directly synthesize enantiopure chiral amines by catalysing the transfer of an amino group from a primary amino donor to a carbonyl acceptor with pyridoxal 5'-phosphate (PLP) as cofactor. In nature, (S)-selective amine transaminases are more abundant than the (R)-selective enzymes, and therefore more information concerning their structures is available. Here, we present the crystal structure of an (R)-ω-transaminase from Aspergillus terreus determined by X-ray crystallography at a resolution of 1.6 Å. The structure of the protein is a homodimer that displays the typical class IV fold of PLP-dependent aminotransferases. The PLP-cofactor observed in the structure is present in two states (i) covalently bound to the active site lysine (the internal aldimine form) and (ii) as substrate/product adduct (the external aldimine form) and free lysine. Docking studies revealed that (R)-transaminases follow a dual binding mode, in which the large binding pocket can harbour the bulky substituent of the amine or ketone substrate and the α-carboxylate of pyruvate or amino acids, and the small binding pocket accommodates the smaller substituent.

Plant lectins, especially those purified from species of the Leguminosae family, represent the best-studied group of carbohydrate-binding proteins. Lectins purified from seeds of the Diocleinae subtribe exhibit a high degree of sequence identity notwithstanding that they show very distinct biological activities. Two main factors have been related to this feature: variance in key residues influencing the carbohydrate-binding site geometry and differences in the pH-dependent oligomeric state profile. In this work, we have isolated a lectin from Canavalia boliviana (Cbol) and solved its x-ray crystal structure in the unbound form and in complex with the carbohydrates Man(α1-3)Man(α1-O)Me, Man(α1-4)Man(α1-O)Me and 5-bromo-4-chloro-3-indolyl-α-D-mannose. We evaluated its oligomerization profile at different pH values using Small Angle X-ray Scattering and compared it to that of Concanavalin A. Based on predicted pKa-shifts of amino acids in the subunit interfaces we devised a model for the dimer-tetramer equilibrium phenomena of these proteins. Additionally, we demonstrated Cbol anti-inflammatory properties and further characterized them using in vivo and in vitro models.

Proline-specific dipeptidyl peptidases (DPPs) are emerging targets for drug development. DPP4 inhibitors are approved in many countries, and other dipeptidyl peptidases are often referred to as DPP4 activity- and/or structure-homologues (DASH). Members of the DASH family have overlapping substrate specificities, and, even though they share low sequence identity, therapeutic or clinical cross-reactivity is a concern. Here, we report the structure of human DPP7 and its complex with a selective inhibitor Dab-Pip (L-2,4-diaminobutyryl-piperidinamide) and compare it with that of DPP4. Both enzymes share a common catalytic domain (α/β-hydrolase). The catalytic pocket is located in the interior of DPP7, deep inside the cleft between the two domains. Substrates might access the active site via a narrow tunnel. The DPP7 catalytic triad is completely conserved and comprises Ser162, Asp418 and His443 (corresponding to Ser630, Asp708 and His740 in DPP4), while other residues lining the catalytic pockets differ considerably. The "specificity domains" are structurally also completely different exhibiting a β-propeller fold in DPP4 compared to a rare, completely helical fold in DPP7. Comparing the structures of DPP7 and DPP4 allows the design of specific inhibitors and thus the development of less cross-reactive drugs. Furthermore, the reported DPP7 structures shed some light onto the evolutionary relationship of prolyl-specific peptidases through the analysis of the architectural organization of their domains.