Proteins-Structure Function and Bioinformatics最新文献

Histone proteins are key players in chromatin packaging. In eukaryotes, nucleosomal cores-the DNA packaging fundamental units-are formed by composition of histone dimers. The double histone fold is a protein structure where two consecutive regions, each featuring histone fold, come together to create a histone pseudodimer. Although regarded as an uncommon fold to date, in this study we show-by protein structure and sequence analyses-that the double histone fold is widespread in eukaryotes. Perspectives of such outcome are discussed in terms of novel directions that our results may open in diverse areas, from epigenetics to the design of DNA-binding proteins.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. The emergence of Mtb's multidrug-resistant and extremely drug-resistant strains has imposed a great challenge for TB treatment. Hence, there is always a demand to explore new targets that may be crucial for the survival and pathogenicity of the bacilli. Oxidoreductases are a class of enzymes that transfer electrons in various biological pathways and reactions, at the expense of cellular NADPH/NADH. Here, we analyzed oxidoreductases from the H37Rv proteome and identified two uncharacterized putative oxidoreductases, Rv1260 and Rv1714. These putative oxidoreductases showed conservation among pathogenic and opportunistic mycobacterial species and were predicted to be virulence factors essential for the pathogen's survival. The 3D structural model and amino acid sequence of one of the oxidoreductases, Rv1260, showed similarities with tetracycline destructase, a flavin-dependent monooxygenase. Thin-layer chromatography and UV-visible spectroscopic experiments confirmed the presence of the FAD molecule in a bound form with the recombinant protein. Fluorescence quenching studies demonstrated a comparatively better affinity of NADPH than NADH with the protein. The protein also displayed efficient binding with chlortetracycline. Molecular dynamics simulations were employed to gain insights into the substrate binding and conformational changes in the protein. Moreover, the importance of the substrate binding region, the C-terminal helix, and the FAD binding cavity, located near the isoalloxazine ring, was highlighted. Overall, the study provides biochemical, biophysical, and mechanistic insights into one of the putative Mtb oxidoreductases. Based on our data, we propose that this protein may perform monooxygenation functions under specific redox conditions and contribute to the redox processes in Mtb.

Homer proteins are modular scaffold molecules that constitute an integral part of the protein network within the postsynaptic density. Full-length Homer1 forms a large homotetramer via a long coiled coil region, and can interact with proline-rich target sequences with its globular EVH1 domain. Here we report an atomistic model of the Homer1 coiled coil region along with the NMR solution structure and backbone dynamics of its EVH1 domain, with implications for the organization of the full-length tetramer. Compared to the already available EVH1 structures, our NMR ensemble exhibits subtle differences, mostly in and around its partner binding region, suggesting the presence of ligand-induced conformational transitions. Molecular dynamics simulations of the long coiled coil reveal distinct regions with different stability and flexibility, with the N-terminal part of the coiled coil exhibiting the largest motions. Interestingly, this segment is highly conserved, pointing to the functional relevance of the observed dynamical features. Our results indicate previously unexplored aspects of the flexibility of the full-length Homer1 tetramer that might contribute to the dynamic rearrangements of the postsynaptic protein network linked to its functional transitions.

Starch is the major energy storage compound in plants. It accumulates in the form of insoluble, partly crystalline granules whose number and shape are specific to each plant species. These characteristics are defined very early in starch biosynthesis, at the initiation stage. Starch biosynthesis initiation is a complex process that relies on the coordinated action of several proteins that interact together in the so-called complex of initiation. Starch Synthase 4 (SS4) is the only initiation protein with enzymatic activity. It catalyzes the formation of glucan primers, which serve as substrates for the enzymatic machinery that synthesizes starch granules. Previous studies have highlighted the importance of interactions between SS4 and regulatory proteins in this process. Among them, Protein Involved in Initiation 1 (PII1) interacts with SS4 but its function is not yet established. In this study, we explored the structural and functional implications of PII1 on SS4's enzymatic activity. Our findings reveal that PII1 contains a long coiled-coil domain that specifically interacts with SS4, leading to modification of SS4's glucan elongation activity. Importantly, this interaction is specific to SS4 and does not affect other known synthases, suggesting a targeted regulatory mechanism probably through a dimerization domain. This work describes the structural specificities of PII1 and SS4 and reveals a possible function for PII1 in the initiation complex.

Anaerobic ammonium-oxidizing (anammox) bacteria employ a unique, hydrazine-based pathway to obtain energy from nitrite and ammonium. These organisms possess distinct Rieske/cytochrome b complexes whose precise role in anammox metabolism remains unclear, but which have been proposed to include the generation of NAD(P)H. This would require energetics and structural features unusual for such complexes. Here we present crystal structures and electrochemical investigations of the Rieske subunits of two of these complexes from the anammox organism Kuenenia stuttgartiensis, Kuste4569 and Kustd1480. Both proteins display high redox potentials (> + 300 mV), which can be in part explained by their crystal structures and which fit perfectly into the energetic scheme of the proposed NAD(P)H generation mechanism. Moreover, AlphaFold3 models of the parent complexes trace out a path for the electrons required for NAD(P)H production, which includes a proposed, novel b-type heme in the membrane-bound part of the complex.

Human citrate synthase (hCS) is a mitochondrial enzyme that catalyzes the aldol condensation of acetyl coenzyme A (AcCoA) to oxaloacetate to form citrate in the TCA cycle. CS activity is important for aerobic exercise performance and basic metabolic function as a housekeeping enzyme. It has been shown through several mass spectrometry-based physiological studies that CS is post-translationally modified (PTM) on numerous residues via acetylation, phosphorylation, and methylation reactions. Few follow-up studies have been reported on the impact of PTMs on CS activity. Thus, we kinetically characterized several hCS PTM mimics near and distant from the active site by site-directed mutagenesis coupled with steady-state kinetics. Most modifications had a negative impact on AcCoA k_cat/K_m but to a much lesser extent on oxaloacetate k_cat/K_m. Most notably, the K393 acetylation mimic, K393Q displays an increase in K_m for AcCoA relative to WT by about 30-fold, with no significant change in k_cat. To complement our kinetic analyses, we performed molecular dynamics simulations on 26 PTM and mutant CS-substrate complexes, providing a combined kinetic and MD simulation approach. Among the MD results, CS K393AcK showed the greatest reduction in AcCoA/CoA binding.

Galectins are a family of carbohydrate-binding proteins that aid in the progression of pathological conditions such as inflammation, bone disease, and cancers, making them attractive drug targets. Galectins share a conserved carbohydrate recognition domain providing a focus for inhibitor design incorporating modification of carbohydrate-based scaffolds that includes the addition of aromatic moieties. Some compounds exhibit nanomolar affinities but often show poor specificity toward particular galectins; thus cross-reactivity in the body is difficult to avoid and potentially detrimental for drug therapies. The low selectivity is due to the high conservation of amino acid residues among galectins that partake in ligand binding. Critically, although these amino acids are highly conserved, there are differences in the shape and physical characteristics of the binding site due to the slight variation in the surrounding amino acid residues of the more extended regions. Using molecular dynamic simulations, the effect of amino acids associated with the galectin binding site was explored. Besides the impact of large bulky side chain amino acids such as phenylalanine, tyrosine, and histidine, a series of salt-bridge interactions were identified within some galectins that influence the resulting shape of the binding site cavity, thus affecting ligand selectivity. In silico alanine point mutations disrupting these salt-bridge interactions or replacing the bulky side chain amino acid residues led to changes in the binding site conformation impacting ligand binding, as indicated by our cluster and energetic analyses. This investigation gives further insight into the galectin carbohydrate binding site that has relevance for ligand design as potential therapeutics.

The generation of antibodies against integral membrane proteins (IMPs) presents unique challenges due to IMPs' low natural abundance, reduced biosynthesis rate, limited level of extraction, and low purification yield. That stems from their intricate structure defining the conformal epitope location. This study introduces a novel approach to identify crucial amino acid residues involved in the interaction between antibodies and tumor-associated transmembrane antigens based on utilizing a prokaryotic cell-free expression (CFPE) system. We considered human transmembrane prostate androgen-induced protein 1 (hTMEPA1) as the target antigen. It is a single-pass α-helical protein possessing hallmarks of a promising cancer biomarker. A several-step procedure was implemented to determine key residues of hTMEPA1. We created mimotope mAbs based on in silico analysis of immunogenic regions followed by target protein antigen production in the CFPE system and further antibody characterization. The results demonstrated an exceptional way for robust and high-throughput target protein production combined with in-depth biophysical and immunochemical characterization of therapeutic and diagnostic antibody-based molecules.

Majority of the proteome is constituted by oligomers and their function is governed by underlying protein-protein interactions. Interfacial residues, namely residues right at the interface of two protein chains, are known to confer stability and specificity in dimers. However, other interactions play a significant role in the formation and maintenance of oligomers in protein assemblies as well. Inter-protein bifurcated interactions are those where one residue simultaneously interacts with two residues belonging to two neighboring protein chains. The characteristic features of such higher-order interactions remain largely unexplored and unknown. In this study, we focused on residues specifically involved in bifurcated interactions (referred as RBI). We examine the bifurcated inter-protein interactions by assembling a dataset of protein assemblies of known 3D structures. We have characterized the type of interactions and the residues involved in the interactions using parameters like energy contributions and conservation scores. We find that the residues participating in bifurcated inter-protein interactions contribute more to the stability of the complex than other interfacial residues. Furthermore, we have presented examples where mutation of a residue involved in a bifurcated interaction results in detrimental outcomes. This study highlights the significance of inter-protein bifurcated interactions that contribute to the stability of multiple interfaces in protein oligomers and hence contribute to the expansion of the understanding of protein assemblies.

Erythrose-4-phosphate dehydrogenase (E4PDH, EC 1.2.1.72) from Acinetobacter baumannii (AbE4PDH) is an essential multifunctional enzyme. E4PDH catalyzes the first step of the deoxyxylulose-5-phosphate (DXP) dependent Vitamin B6 biosynthetic pathway. It utilizes nicotinamide adenine diphosphate (NAD⁺) as a co-factor while exhibiting dual catalytic activity wherein it converts (i) D-Erythrose-4-phosphate to 4-phosphoerythronate and (ii) Glyceraldehyde-3-phosphate to Glyceraldehyde-1,3-bisphosphate. An alternate function of AbE4PDH is the capture of human transferrin (Tf) and lactoferrin (Lf) for bacterial iron acquisition. This study provides the first X-ray crystal structures of AbE4PDH at a resolution of 2.2 Å. The recombinant enzyme was crystallized under two different conditions with (i) 30% PEG-400 (Crystal/Structure 1; PDB Id-9IIL) and (ii) 1.6 M MgSO₄ (Crystal/Structure 2; PDB Id-9IIM). The crystal structure reveals a homo-tetramer, with an NAD⁺ bound to each monomer. The structure from Crystal 2 contained only four sulfate ions which were located in the substrate binding sites. In contrast, Crystal 1 showed the presence of several polyethylene glycol (PEG) molecules located in the center of the tetramer contributing to its stability without disturbing co-factor binding. The presence of PEG molecules induced a strong conformational change in the side chain of Gln213, inducing the formation of additional intermolecular contacts and enhancing the stability of the tetramer. Overall, this novel structure has not been observed with other dehydrogenases and may be unique to E4PDH. An insight into this structure would aid in the design of potential small molecule inhibitors to target its biochemical and alternate functions.