Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans.

Loss-of-function Parkin mutations lead to early-onset of Parkinson's disease. Parkin is an auto-inhibited ubiquitin E3 ligase activated by dual phosphorylation of its ubiquitin-like (Ubl) domain and ubiquitin by the PINK1 kinase. Herein, we demonstrate a competitive binding of the phospho-Ubl and RING2 domains towards the RING0 domain, which regulates Parkin activity. We show that phosphorylated Parkin can complex with native Parkin, leading to the activation of autoinhibited native Parkin in trans. Furthermore, we show that the activator element (ACT) of Parkin is required to maintain the enzyme kinetics, and the removal of ACT slows the enzyme catalysis. We also demonstrate that ACT can activate Parkin in trans but less efficiently than when present in the cis molecule. Furthermore, the crystal structure reveals a donor ubiquitin binding pocket in the linker connecting REP and RING2, which plays a crucial role in Parkin activity.

Cryo-EM structures of a mycobacterial ABC transporter that mediates rifampicin resistance.

Drug-resistant Tuberculosis (TB) is a global public health problem. Resistance to rifampicin, the most effective drug for TB treatment, is a major growing concern. The etiological agent, Mycobacterium tuberculosis (Mtb), has a cluster of ATP-binding cassette (ABC) transporters which are responsible for drug resistance through active export. Here, we describe studies characterizing Mtb Rv1217c-1218c as an ABC transporter that can mediate mycobacterial resistance to rifampicin and have determined the cryo-electron microscopy structures of Rv1217c-1218c. The structures show Rv1217c-1218c has a type V exporter fold. In the absence of ATP, Rv1217c-1218c forms a periplasmic gate by two juxtaposed-membrane helices from each transmembrane domain (TMD), while the nucleotide-binding domains (NBDs) form a partially closed dimer which is held together by four salt-bridges. Adenylyl-imidodiphosphate (AMPPNP) binding induces a structural change where the NBDs become further closed to each other, which downstream translates to a closed conformation for the TMDs. AMPPNP binding results in the collapse of the outer leaflet cavity and the opening of the periplasmic gate, which was proposed to play a role in substrate export. The rifampicin-bound structure shows a hydrophobic and periplasm-facing cavity is involved in rifampicin binding. Phospholipid molecules are observed in all determined structures and form an integral part of the Rv1217c-1218c transporter system. Our results provide a structural basis for a mycobacterial ABC exporter that mediates rifampicin resistance, which can lead to different insights into combating rifampicin resistance.

Structural and functional effects of phosphopriming and scaffolding in the kinase GSK-3ß.

Glycogen synthase kinase 3ß (GSK-3ß) targets specific signaling pathways in response to distinct upstream signals. We used structural and functional studies to dissect how an upstream phosphorylation step primes the Wnt signaling component ß-catenin for phosphorylation by GSK-3ß and how scaffolding interactions contribute to this reaction. Our crystal structure of GSK-3ß bound to a phosphoprimed ß-catenin peptide confirmed the expected binding mode of the phosphoprimed residue adjacent to the catalytic site. An aspartate phosphomimic in the priming site of ß-catenin adopted an indistinguishable structure but reacted approximately 1000-fold slower than the native phosphoprimed substrate. This result suggests that substrate positioning alone is not sufficient for catalysis and that native phosphopriming interactions are necessary. We also obtained a structure of GSK-3ß with an extended peptide from the scaffold protein Axin that bound with greater affinity than that of previously crystallized Axin fragments. This structure neither revealed additional contacts that produce the higher affinity nor explained how substrate interactions in the GSK-3ß active site are modulated by remote Axin binding. Together, our findings suggest that phosphopriming and scaffolding produce small conformational changes or allosteric effects, not captured in the crystal structures, that activate GSK-3ß and facilitate ß-catenin phosphorylation. These results highlight limitations in our ability to predict catalytic activity from structure and have potential implications for the role of natural phosphomimic mutations in kinase regulation and phosphosite evolution.

Bacterial ß-carbonic anhydrases.

ß-Carbonic anhydrases (ß-CA; EC 4.2.1.1) are widespread zinc metalloenzymes which catalyze the interconversion of carbon dioxide and bicarbonate. They have been isolated in many pathogenic and non-pathogenic bacteria where they are involved in multiple roles, often related to their growth and survival. ß-CAs are structurally distant from the CAs of other classes. In the active site, located at the interface of a fundamental dimer, the zinc ion is coordinated to two cysteines and one histidine. ß-CAs have been divided in two subgroups depending on the nature of the fourth ligand on the zinc ion: class I have a zinc open configuration with a hydroxide ion completing the metal coordination, which is the catalytically active species in the mechanism proposed for the ß-CAs similar to the well-known of α-CAs, while in class II an Asp residue substitute the hydroxide. This latter active site configuration has been showed to be typical of an inactive form at pH below 8. An Asp-Arg dyad is thought to play a key role in the pH-induced catalytic switch regulating the opening and closing of the active site in class II ß-CAs, by displacing the zinc-bound solvent molecule. An allosteric site well-suited for bicarbonate stabilizes the inactive form. This bicarbonate binding site is composed by a triad of well conserved residues, strictly connected to the coordination state of the zinc ion. Moreover, the escort site is a promiscuous site for a variety of ligands, including bicarbonate, at the dimer interface, which may be the route for bicarbonate to the allosteric site.

Association mechanism of bicalutamide and human serum albumin for potential clinical implications.

The binding mechanism of molecular interaction between bicalutamide and human serum albumin (HSA) in a pH 7.4 phosphate buffer was studied using various spectroscopic techniques in combination with molecular modeling. Fluorescence data revealed that the fluorescence quenching of HSA by bicalutamide was a static quenching procedure. The binding constants and number of binding sites were evaluated at different temperatures. The thermodynamic parameters, ΔH and ΔS, were calculated to be 4.30 × 104 J·mol-1 and 245 J·mol-1·K-1, respectively, suggesting that the binding of bicalutamide to HSA was driven mainly by hydrophobic interactions and hydrogen bonds. The displacement studies indicated neither Sudlow's site I nor II but subdomain IB as the main binding site for bicalutamide on HSA. The binding distance between bicalutamide and HSA was determined to be 3.54 nm based on the Förster theory. Analysis of circular dichroism, synchronous, and 3D fluorescence spectra demonstrated that HSA conformation was slightly altered in the presence of bicalutamide.

Pretrainable geometric graph neural network for antibody affinity maturation.

Increasing the binding affinity of an antibody to its target antigen is a crucial task in antibody therapeutics development. This paper presents a pretrainable geometric graph neural network, GearBind, and explores its potential in in silico affinity maturation. Leveraging multi-relational graph construction, multi-level geometric message passing and contrastive pretraining on mass-scale, unlabeled protein structural data, GearBind outperforms previous state-of-the-art approaches on SKEMPI and an independent test set. A powerful ensemble model based on GearBind is then derived and used to successfully enhance the binding of two antibodies with distinct formats and target antigens. ELISA EC50 values of the designed antibody mutants are decreased by up to 17 fold, and KD values by up to 6.1 fold. These promising results underscore the utility of geometric deep learning and effective pretraining in macromolecule interaction modeling tasks.

​Fusarium Protein Toolkit: a web-based resource for structural and variant analysis of Fusarium species.

BACKGROUND: ​​The genus Fusarium poses significant threats to food security and safety worldwide because numerous species of the fungus cause destructive diseases and/or mycotoxin contamination in crops. The adverse effects of climate change are exacerbating some existing threats and causing new problems. These challenges highlight the need for innovative solutions, including the development of advanced tools to identify targets for control strategies. DESCRIPTION: In response to these challenges, we developed the Fusarium Protein Toolkit (FPT), a web-based tool that allows users to interrogate the structural and variant landscape within the Fusarium pan-genome. The tool displays both AlphaFold and ESMFold-generated protein structure models from six Fusarium species. The structures are accessible through a user-friendly web portal and facilitate comparative analysis, functional annotation inference, and identification of related protein structures. Using a protein language model, FPT predicts the impact of over 270 million coding variants in two of the most agriculturally important species, Fusarium graminearum and F. verticillioides. To facilitate the assessment of naturally occurring genetic variation, FPT provides variant effect scores for proteins in a Fusarium pan-genome based on 22 diverse species. The scores indicate potential functional consequences of amino acid substitutions and are displayed as intuitive heatmaps using the PanEffect framework. CONCLUSION: FPT fills a knowledge gap by providing previously unavailable tools to assess structural and missense variation in proteins produced by Fusarium. FPT has the potential to deepen our understanding of pathogenic mechanisms in Fusarium, and aid the identification of genetic targets for control strategies that reduce crop diseases and mycotoxin contamination. Such targets are vital to solving the agricultural problems incited by Fusarium, particularly evolving threats resulting from climate change. Thus, FPT has the potential to contribute to improving food security and safety worldwide.

Molecular Modeling Methods in the Development of Affine and Specific Protein-Binding Agents.

High-affinity and specific agents are widely applied in various areas, including diagnostics, scientific research, and disease therapy (as drugs and drug delivery systems). It takes significant time to develop them. For this reason, development of high-affinity agents extensively utilizes computer methods at various stages for the analysis and modeling of these molecules. The review describes the main affinity and specific agents, such as monoclonal antibodies and their fragments, antibody mimetics, aptamers, and molecularly imprinted polymers. The methods of their obtaining as well as their main advantages and disadvantages are briefly described, with special attention focused on the molecular modeling methods used for their analysis and development.

Structures of the human leading strand Polε-PCNA holoenzyme.

In eukaryotes, the leading strand DNA is synthesized by Polε and the lagging strand by Polδ. These replicative polymerases have higher processivity when paired with the DNA clamp PCNA. While the structure of the yeast Polε catalytic domain has been determined, how Polε interacts with PCNA is unknown in any eukaryote, human or yeast. Here we report two cryo-EM structures of human Polε-PCNA-DNA complex, one in an incoming nucleotide bound state and the other in a nucleotide exchange state. The structures reveal an unexpected three-point interface between the Polε catalytic domain and PCNA, with the conserved PIP (PCNA interacting peptide)-motif, the unique P-domain, and the thumb domain each interacting with a different protomer of the PCNA trimer. We propose that the multi-point interface prevents other PIP-containing factors from recruiting to PCNA while PCNA functions with Polε. Comparison of the two states reveals that the finger domain pivots around the [4Fe-4S] cluster-containing tip of the P-domain to regulate nucleotide exchange and incoming nucleotide binding.

The structural basis for the collagen processing by human P3H1/CRTAP/PPIB ternary complex.

Collagen posttranslational processing is crucial for its proper assembly and function. Disruption of collagen processing leads to tissue development and structure disorders like osteogenesis imperfecta (OI). OI-related collagen processing machinery includes prolyl 3-hydroxylase 1 (P3H1), peptidyl-prolyl cis-trans isomerase B (PPIB), and cartilage-associated protein (CRTAP), with their structural organization and mechanism unclear. We determine cryo-EM structures of the P3H1/CRTAP/PPIB complex. The active sites of P3H1 and PPIB form a face-to-face bifunctional reaction center, indicating a coupled modification mechanism. The structure of the P3H1/CRTAP/PPIB/collagen peptide complex reveals multiple binding sites, suggesting a substrate interacting zone. Unexpectedly, a dual-ternary complex is observed, and the balance between ternary and dual-ternary states can be altered by mutations in the P3H1/PPIB active site and the addition of PPIB inhibitors. These findings provide insights into the structural basis of collagen processing by P3H1/CRTAP/PPIB and the molecular pathology of collagen-related disorders.

Structural basis of eukaryotic transcription termination by the Rat1 exonuclease complex.

The 5´-3´ exoribonuclease Rat1/Xrn2 is responsible for the termination of eukaryotic mRNA transcription by RNAPII. Rat1 forms a complex with its partner proteins, Rai1 and Rtt103, and acts as a "torpedo" to bind transcribing RNAPII and dissociate DNA/RNA from it. Here we report the cryo-electron microscopy structures of the Rat1-Rai1-Rtt103 complex and three Rat1-Rai1-associated RNAPII complexes (type-1, type-1b, and type-2) from the yeast, Komagataella phaffii. The Rat1-Rai1-Rtt103 structure revealed that Rat1 and Rai1 form a heterotetramer with a single Rtt103 bound between two Rai1 molecules. In the type-1 complex, Rat1-Rai1 forms a heterodimer and binds to the RNA exit site of RNAPII to extract RNA into the Rat1 exonuclease active site. This interaction changes the RNA path in favor of termination (the "pre-termination" state). The type-1b and type-2 complexes have no bound DNA/RNA, likely representing the "post-termination" states. These structures illustrate the termination mechanism of eukaryotic mRNA transcription.

Exploring the structural landscape of DNA maintenance proteins.

Evolutionary annotation of genome maintenance (GM) proteins has conventionally been established by remote relationships within protein sequence databases. However, often no significant relationship can be established. Highly sensitive approaches to attain remote homologies based on iterative profile-to-profile methods have been developed. Still, these methods have not been systematically applied in the evolutionary annotation of GM proteins. Here, by applying profile-to-profile models, we systematically survey the repertoire of GM proteins from bacteria to man. We identify multiple GM protein candidates and annotate domains in numerous established GM proteins, among other PARP, OB-fold, Macro, TUDOR, SAP, BRCT, KU, MYB (SANT), and nuclease domains. We experimentally validate OB-fold and MIS18 (Yippee) domains in SPIDR and FAM72 protein families, respectively. Our results indicate that, surprisingly, despite the immense interest and long-term research efforts, the repertoire of genome stability caretakers is still not fully appreciated.

Unveiling the solution structure of a DNA duplex with continuous silver-modified Watson-Crick base pairs.

The challenge of transforming organized DNA structures into their metallized counterparts persists in the scientific field. In this context, utilizing DNA molecules modified with 7-deazapurine, provides a transformative solution. In this study, we present the solution structure of a DNA duplex that can be transformed into its metallized equivalent while retaining the natural base pairing arrangement through the creation of silver-modified Watson-Crick base pairs. Unlike previously documented X-ray structures, our research demonstrates the feasibility of preserving the intrinsic DNA self-assembly while incorporating AgI into the double helix, illustrating that the binding of silver does not disrupt the canonical base-pairing organization. Moreover, in our case, the uninterrupted AgI chain deviates from forming conventional straight linear chains; instead, it adheres to a helical arrangement dictated by the underlying DNA structure. This research challenges conventional assumptions and opens the door to precisely design structures based on the organization of highly stable Ag-DNA assemblies.

A bitopic agonist bound to the dopamine 3 receptor reveals a selectivity site.

Although aminergic GPCRs are the target for ~25% of approved drugs, developing subtype selective drugs is a major challenge due to the high sequence conservation at their orthosteric binding site. Bitopic ligands are covalently joined orthosteric and allosteric pharmacophores with the potential to boost receptor selectivity and improve current medications by reducing off-target side effects. However, the lack of structural information on their binding mode impedes rational design. Here we determine the cryo-EM structure of the hD3R:GαOßγ complex bound to the D3R selective bitopic agonist FOB02-04A. Structural, functional and computational analyses provide insights into its binding mode and point to a new TM2-ECL1-TM1 region, which requires the N-terminal ordering of TM1, as a major determinant of subtype selectivity in aminergic GPCRs. This region is underexploited in drug development, expands the established secondary binding pocket in aminergic GPCRs and could potentially be used to design novel and subtype selective drugs.

Structural Basis for Evasion of New SARS-CoV-2 Variants from the Potent Virus-Neutralizing Nanobody Targeting the S-Protein Receptor-Binding Domain.

COVID-19 has caused millions of deaths and many times more infections worldwide, emphasizing the unpreparedness of the global health system in the face of new infections and the key role for vaccines and therapeutics, including virus-neutralizing antibodies, in prevention and containment of the disease. Continuous evolution of the SARS-CoV-2 coronavirus has been causing its new variants to evade the action of the immune system, which highlighted the importance of detailed knowledge of the epitopes of already selected potent virus-neutralizing antibodies. A single-chain antibody ("nanobody") targeting the SARS-CoV-2 receptor-binding domain (RBD), clone P2C5, had exhibited robust virus-neutralizing activity against all SARS-CoV-2 variants and, being a major component of the anti-COVID-19 formulation "GamCoviMab", had successfully passed Phase I of clinical trials. However, after the emergence of the Delta and XBB variants, a decrease in the neutralizing activity of this nanobody was observed. Here we report on the successful crystal structure determination of the RBD:P2C5 complex at 3.1 Å, which revealed the intricate protein-protein interface, sterically occluding full ACE2 receptor binding by the P2C5-neutralized RBD. Moreover, the structure revealed the developed RBD:P2C5 interface centered around residues Leu452 and Phe490, thereby explaining the evasion of the Delta or Omicron XBB, but not Omicron B.1.1.529 variant, as a result of the single L452R or F490S mutations, respectively, from the action of P2C5. The structure obtained is expected to foster nanobody engineering in order to rescue neutralization activity and will facilitate epitope mapping for other neutralizing nanobodies by competition assays.

Mechanism of PARP1 Elongation Reaction Revealed by Molecular Modeling.

Poly(ADP-ribose) polymerase 1 (PARP1) plays a major role in the DNA damage repair and transcriptional regulation, and is targeted by a number of clinical inhibitors. Despite this, catalytic mechanism of PARP1 remains largely underexplored because of the complex substrate/product structure. Using molecular modeling and metadynamics simulations we have described in detail elongation of poly(ADP-ribose) chain in the PARP1 active site. It was shown that elongation reaction proceeds via the SN1-like mechanism involving formation of the intermediate furanosyl oxocarbenium ion. Intriguingly, nucleophilic 2'A-OH group of the acceptor substrate can be activated by the general base Glu988 not directly but through the proton relay system including the adjacent 3'A-OH group.

Allosteric modulation of the CXCR4:CXCL12 axis by targeting receptor nanoclustering via the TMV-TMVI domain.

CXCR4 is a ubiquitously expressed chemokine receptor that regulates leukocyte trafficking and arrest in both homeostatic and pathological states. It also participates in organogenesis, HIV-1 infection, and tumor development. Despite the potential therapeutic benefit of CXCR4 antagonists, only one, plerixafor (AMD3100), which blocks the ligand-binding site, has reached the clinic. Recent advances in imaging and biophysical techniques have provided a richer understanding of the membrane organization and dynamics of this receptor. Activation of CXCR4 by CXCL12 reduces the number of CXCR4 monomers/dimers at the cell membrane and increases the formation of large nanoclusters, which are largely immobile and are required for correct cell orientation to chemoattractant gradients. Mechanistically, CXCR4 activation involves a structural motif defined by residues in TMV and TMVI. Using this structural motif as a template, we performed in silico molecular modeling followed by in vitro screening of a small compound library to identify negative allosteric modulators of CXCR4 that do not affect CXCL12 binding. We identified AGR1.137, a small molecule that abolishes CXCL12-mediated receptor nanoclustering and dynamics and blocks the ability of cells to sense CXCL12 gradients both in vitro and in vivo while preserving ligand binding and receptor internalization.

Advanced vaccinomic, immunoinformatic, and molecular modeling strategies for designing Multi- epitope vaccines against the Enterobacter cloacae complex.

The increasing and ongoing issue of antibiotic resistance in bacteria is of huge concern globally, mainly to healthcare facilities. It is now crucial to develop a vaccine for therapeutic and preventive purposes against the bacterial species causing hospital-based infections. Among the many antibiotic- resistant bacterial pathogens, the Enterobacter cloacae complex (ECC) including six species, E. Colcae, E. absuriae, E. kobie, E. hormaechei, E. ludwigii, and E. nimipressuralis, are dangerous to public health and may worsen the situation. Vaccination plays a vital role in the prevention of infections and infectious diseases. This research highlighted the construction and design of a multi-epitope vaccine for the E. cloacae complex by retrieving their complete sequenced proteome. The retrieved proteome was assessed to opt for potential vaccine candidates using immunoinformatic tools. Both B and T-cell epitopes were predicted in order to create both humoral and cellular immunity and further scrutinized for antigenicity, allergenicity, water solubility, and toxicity analysis. The final potential epitopes were subjected to population coverage analysis. Major histocompatibility complex (MHC) class combined, and MHC Class I and II world population coverage was obtained as 99.74%, and 98.55% respectively while a combined 81.81% was covered. A multi-epitope peptide-based vaccine construct consisting of the adjuvant, epitopes, and linkers was subjected to the ProtParam tool to calculate its physiochemical properties. The total amino acids were 236, the molecular weight was 27.64kd, and the vaccine construct was stable with an instability index of 27.01. The Grand Average of Hydropathy (GRAVY) (hydrophilicity) value obtained was -0.659, being more negative and depicting the hydrophilic character. It was non-allergen antigenic with an antigenicity of 0.8913. The vaccine construct was further validated for binding efficacy with immune cell receptors MHC-I, MHC-II, and Toll-like receptor (TLR)-4. The molecular docking results depict that the designed vaccine has good binding potency with immune receptors crucial for antigen presentation and processing. Among the Vaccine-MHC-I, Vaccine-MHC-II, and Vaccine-TLR-4 complexes, the best-docked poses were identified based on their lowest binding energy scores of -886.8, -995.6, and -883.6, respectively. Overall, we observed that the designed vaccine construct can evoke a proper immune response and the construct could help experimental researchers in the formulation of a vaccine against the targeted pathogens.

Insights into the structure and activation mechanism of some class B1 GPCR family members.

In humans, 15 genes encode the class B1 family of GPCRs, which are polypeptide hormone receptors characterized by having a large N-terminal extracellular domain (ECD) and receive signals from outside the cell to activate cellular response. For example, the insulinotropic polypeptide (GIP) stimulates the glucose-dependent insulinotropic polypeptide receptor (GIPR), while the glucagon receptor (GCGR) responds to glucagon by increasing blood glucose levels and promoting the breakdown of liver glycogen to induce the production of insulin. The glucagon-like peptides 1 and 2 (GLP-1 and GLP-2) elicit a response from glucagon-like peptide receptor types 1 and 2 (GLP1R and GLP2R), respectively. Since these receptors are implicated in the pathogenesis of diabetes, studying their activation is crucial for the development of effective therapies for the condition. With more structural information being revealed by experimental methods such as X-ray crystallography, cryo-EM, and NMR, the activation mechanism of class B1 GPCRs becomes unraveled. The available crystal and cryo-EM structures reveal that class B1 GPCRs follow a two-step model for peptide binding and receptor activation. The regions close to the C-termini of hormones interact with the N-terminal ECD of the receptor while the regions close to the N-terminus of the peptide interact with the TM domain and transmit signals. This review highlights the structural details of class B1 GPCRs and their conformational changes following activation. The roles of MD simulation in characterizing those conformational changes are briefly discussed, providing insights into the potential structural exploration for future ligand designs.

Decoding the molecular mechanism of selective autophagy of glycogen mediated by autophagy receptor STBD1.

Autophagy of glycogen (glycophagy) is crucial for the maintenance of cellular glucose homeostasis and physiology in mammals. STBD1 can serve as an autophagy receptor to mediate glycophagy by specifically recognizing glycogen and relevant key autophagic factors, but with poorly understood mechanisms. Here, we systematically characterize the interactions of STBD1 with glycogen and related saccharides, and determine the crystal structure of the STBD1 CBM20 domain with maltotetraose, uncovering a unique binding mode involving two different oligosaccharide-binding sites adopted by STBD1 CBM20 for recognizing glycogen. In addition, we demonstrate that the LC3-interacting region (LIR) motif of STBD1 can selectively bind to six mammalian ATG8 family members. We elucidate the detailed molecular mechanism underlying the selective interactions of STBD1 with ATG8 family proteins by solving the STBD1 LIR/GABARAPL1 complex structure. Importantly, our cell-based assays reveal that both the STBD1 LIR/GABARAPL1 interaction and the intact two oligosaccharide binding sites of STBD1 CBM20 are essential for the effective association of STBD1, GABARAPL1, and glycogen in cells. Finally, through mass spectrometry, biochemical, and structural modeling analyses, we unveil that STBD1 can directly bind to the Claw domain of RB1CC1 through its LIR, thereby recruiting the key autophagy initiation factor RB1CC1. In all, our findings provide mechanistic insights into the recognitions of glycogen, ATG8 family proteins, and RB1CC1 by STBD1 and shed light on the potential working mechanism of STBD1-mediated glycophagy.