Protein Science最新文献

The point mutation N642H of the signal transducer and activator of transcription 5B (STAT5B) protein is associated with aggressive and drug-resistant forms of leukemia. This mutation is thought to promote cancer due to hyperactivation of STAT5B caused by increased stability of the active, parallel dimer state. However, the molecular mechanism leading to this stabilization is not well understood as there is currently no structure of the parallel dimer. To investigate the mutation's mechanism of action, we conducted extensive all-atom molecular dynamics simulations of multiple oligomeric forms of both STAT5B and STAT5B^N642H, including a model for the parallel dimer. The N642H mutation directly affects the hydrogen bonding network within the phosphotyrosine (pY)-binding pocket of the parallel dimer, enhancing the pY-binding interaction. The simulations indicate that apo STAT5B is highly flexible, exploring a diverse conformational space. In contrast, apo STAT5B^N642H accesses two distinct conformational states, one of which resembles the conformation of the parallel dimer. The simulation predictions of the effects of the mutation on structure and dynamics are supported by the results of hydrogen-deuterium exchange (HDX) mass spectrometry measurements carried out on STAT5B and STAT5B^N642H in which a phosphopeptide was used to mimic the effects of parallel dimerization on the SH2 domain. The molecular-level information uncovered in this work contributes to our understanding of STAT5B hyperactivation by the N642H mutation and could help pave the way for novel therapeutic strategies targeting this mutation.

Peptides are attracting a growing interest for therapeutic applications in biomedicine. In Parkinson's disease (PD), different human endogenous peptides have been associated with beneficial effects, including protein aggregation inhibition, reduced inflammation, or the protection of dopaminergic neurons. Such effects seem to be connected to the spatial arrangement of peptide side chains, and many of these human molecules share common conformational traits, displaying a distinctive amphipathic and cationic helical structure, which is believed to be crucial for their activities. This review delves into the relationship between these structural properties and the current evidence connecting biogenic peptides to the amelioration of PD symptoms. We discuss their implications in the disease, the different mechanisms of action, their state of validation, and their therapeutic potential.

Antimicrobial resistance is a significant cause of mortality globally due to infections, a trend that is expected to continue to rise. As existing treatments fail and new drug discovery slows, the urgency to develop novel antimicrobial therapeutics grows stronger. One promising strategy involves targeting bacterial systems exclusive to pathogens, such as the transcription regulator protein GabR. Expressed in diverse bacteria including Escherichia coli, Bordetella pertussis, and Klebsiella pneumoniae, GabR has no homolog in eukaryotes, making it an ideal therapeutic target. Bacillus subtilis GabR (bsGabR), the most studied variant, regulates its own transcription and activates genes for GABA aminotransferase (GabT) and succinic semialdehyde dehydrogenase (GabD). This intricate regulatory system presents a compelling antimicrobial target with the potential for agonistic intervention to disrupt bacterial gene expression and induce cellular dysfunction, especially in bacterial stress responses. To explore manipulation of this system and the potential of this protein as an antimicrobial target, an in-depth understanding of the unique PLP-dependent transcription regulation is critical. Herein, we report the successful structural modification of the cofactor PLP and demonstrate the biochemical reactivity of the PLP analog pyridoxal-5'-tetrazole (PLT). Through both spectrophotometric and X-ray crystallographic analyses, we explore the interaction between bsGabR and PLT, together with a synthesized GABA derivative (S)-4-amino-5-phenoxypentanoate (4-phenoxymethyl-GABA or 4PMG). Most notably, we present a crystal structure of the condensed, external aldimine complex within bsGabR. While PLT alone is not a drug candidate, it can act as a probe to study the detailed mechanism of GabR-mediated function. PLT employs a tetrazole moiety as a bioisosteric replacement for phosphate in PLP. In addition, the PLP-4PMG adduct observed in the structure may serve as a novel chemical scaffold for subsequent structure-based antimicrobial design.

After overexpression in a suitable host, recombinant protein purification often relies on affinity (e.g., poly-histidine) and solubility-enhancing (e.g., small ubiquitin-like-modifier [SUMO]) tags. Following purification, these tags are removed to avoid their interference with target protein structure and function. The wide use of N-terminal His₆-SUMO fusions is partly due to efficient cleavage of the SUMO tag's C-terminal Gly-Gly motif by the Ulp1 SUMO protease and generation of the native N-terminus of the target protein. While adopting this system to purify the Salmonella homodimeric FraB deglycase, we discovered that Shine-Dalgarno (SD) sequences in the eukaryotic SUMO tag resulted in truncated proteins. This finding has precedents for synthesis of partial proteins in Escherichia coli from cryptic ribosome-binding sites within eukaryotic coding sequences. The SUMO open reading frame has two "GGNGGN" motifs that resemble SD sequences, one of which encodes the Gly-Gly motif required for Ulp1 cleavage. By mutating these SD sequences, we generated SUMO^NIT (no internal translation), a variant that eliminated production of the truncated proteins without affecting the levels of full-length His₆-SUMO-FraB or Ulp1 cleavage. SUMO^NIT should be part of the toolkit for enhancing SUMO fusion protein yield, purity, and homogeneity (especially for homo-oligomers). Moreover, we showcase the value of native mass spectrometry in revealing the complications that arise from generation of truncated proteins, as well as oxidation events and protease inhibitor adducts, which are indiscernible by commonly employed lower resolution methods.

Antibodies and antibody mimics are extensively used in the pharmaceutical industry, where stringent safety standards are required. Implementing heat sterilization during or after the manufacturing process could help prevent contamination by viruses and bacteria. However, conventional antibodies and antibody mimics are not suitable for heat sterilization because they irreversibly denature at high temperatures. In this study, we focused on the refolding property of the cold shock protein from the hyperthermophile Thermotoga maritima (TmCSP), which denatures at elevated temperatures but regains its native structure upon re-cooling. We designed and constructed a mutant library of TmCSP in which amino acid residues in its three surface loops were diversified. From the library, mutant TmCSPs that bind to each of eight target proteins were selected by phage and yeast surface display methods. We confirmed that the secondary structure and binding affinity of all the selected mutants were restored after heat treatment followed by cooling. Additionally, freeze-drying did not impair their binding affinity. The crystal structure of a mutant TmCSP in complex with its target, the esterase from Alicyclobacillus acidocaldarius, revealed specific interactions between them. These results clearly demonstrate the feasibility of creating heat-sterilizable antibody mimics using TmCSP as a scaffold.

Intrinsically disordered proteins (IDPs) are important drug targets as they are key actors within cell signaling networks. However, the conformational plasticity of IDPs renders them challenging to characterize, which is a bottleneck in developing small molecule drugs that bind to IDPs and modulate their behavior. In relation to this, ion mobility mass spectrometry (IM-MS) is a useful tool to investigate IDPs, as it can reveal their conformational preferences. It can also offer important insights in drug discovery, as it can measure binding stoichiometry and unveil conformational shifts of IDPs exerted by the binding of small drug-like molecules. Herein, we have used IM-MS to investigate the effect of drug lead EPI-001 on the disordered N-terminal domain of the androgen receptor (AR-NTD). Despite structural heterogeneity rendering the NTD a challenging region of the protein to drug, this domain harbors most, if not all, of the transcriptional activity. We quantify the stoichiometry of EPI-001 binding to various constructs corresponding to functional domains of AR-NTD and show that it binds to separate constructs containing transactivation unit (TAU)-1 and TAU-5, respectively, and that 1-2 molecules bind to a larger construct containing both sequences. We also identify a conformational shift upon EPI-001 binding to the TAU-5, and to a much lesser extent with TAU-1 containing constructs. This work provides novel insight on the interactions of EPI-001 with the AR-NTD, and the structural alterations that it exerts, and positions IM-MS as an informative tool that will enhance the tractability of IDPs, potentially leading to better therapies.

Intracellular lipid binding proteins (iLBPs) play crucial roles in lipid transport and cellular metabolism across the animal kingdom. Recently, a fat-to-neuron axis was described in Caenorhabditis elegans, in which lysosomal activity in the fat liberates polyunsaturated fatty acids (PUFAs) that signal to neurons and extend lifespan with durable fecundity. In this study, we investigate the structure and binding mechanisms of a lifespan-extending lipid chaperone, lipid binding protein-3 (LBP-3), which shuttles dihomo-γ-linolenic (DGLA) acid from intestinal fat to neurons. We present the first high-resolution crystal structure of LBP-3, which reveals a classic iLBP fold with an unexpected and unique homodimeric arrangement via interstrand interactions that is incompatible with ligand binding. We identify key ionic interactions that mediate DGLA binding within the lipid binding pocket. Molecular dynamics simulations further elucidate LBP-3's preferential binding to DGLA due to its rotational freedom and access to favorable binding conformations compared to other 20-carbon PUFAs. We also propose that LBP-3 dimerization may be a unique regulatory mechanism for lipid chaperones.

Human succinic semialdehyde dehydrogenase is a mitochondrial enzyme fundamental in the neurotransmitter γ-aminobutyric acid catabolism. It catalyzes the NAD⁺-dependent oxidative degradation of its derivative, succinic semialdehyde, to succinic acid. Mutations in its gene lead to an inherited neurometabolic rare disease, succinic semialdehyde dehydrogenase deficiency, characterized by mental and developmental delay. Due to the poor characterization of this enzyme, we carried out evolutionary and kinetic investigations to contribute to its functional behavior, a prerequisite to interpreting pathogenic variants. An in silico analysis shows that succinic semialdehyde dehydrogenases belong to two families, one human-like and the other of bacterial origin, differing in the oligomeric state and in a network of active site residues. This information is coupled to the biophysical-biochemical characterization of the human recombinant enzyme uncovering that (i) catalysis proceeds by an ordered bi-bi mechanism with NAD⁺ binding before the aldehyde that exerts a partial non-competitive inhibition; (ii) a stabilizing complex between the catalytic Cys340 and NAD⁺ is observed and interpreted as a protective mechanism; and (iii) a concerted non-covalent network assists the action of the catalytic residues Cys340 and Glu306. Through mutational analyses of Lys214, Glu306, Cys340, and Glu515 associated with pH studies, we showed that NAD⁺ binding is controlled by the dyad Lys214-Glu515. Moreover, catalysis is assured by proton transfer exerted by the same dyad networked with the catalytic Glu306, involved in catalytic Cys340 deprotonation/reprotonation. The identification of this weak bond network essential for cofactor binding and catalysis represents a first step to tackling the molecular basis for its deficiency.

This study focuses on spastic paraplegia type 50 (SPG50), an adapter protein complex 4 deficiency syndrome caused by mutations in the adapter protein complex 4 subunit mu-1 (AP4M1) gene, and on the downstream alterations of the AP4M1 protein. We applied a battery of heterogeneous computational resources, encompassing two in-house tools described here for the first time, to (a) assess the druggability potential of AP4M1, (b) characterize SPG50-associated mutations and their 3D scenario, (c) identify mutation-tailored drug candidates for SPG50, and (d) elucidate their mechanisms of action by means of structural considerations on homology models of the adapter protein complex 4 core. Altogether, the collected results indicate R367Q as the mutation with the most promising potential of being corrected by small-molecule drugs, and the flavonoid rutin as best candidate for this purpose. Rutin shows promise in rescuing the interaction between the AP4M1 and adapter protein complex subunit beta-1 (AP4B1) subunits by means of a glue-like mode of action. Overall, this approach offers a framework that could be systematically applied to the investigation of mutation-wise molecular mechanisms in different hereditary spastic paraplegias, too.

Enzyme stability can be measured in a number of ways, including melting temperature, activity retention, and size analysis. However, these measurements are often conducted in an idealized storage buffer and not in the relevant enzymatic reaction media. Particularly for reactions that occur in alkaline, volatile, and high ionic strength media, typical analyses using differential scanning calorimetry, light scattering, and sodium dodecyl-sulfate polyacrylamide gel electrophoresis are not satisfactory to track the stability of these enzymes. In this work, we monitor the stability of engineered and native dehydrogenases that require a high amount of ammonia for their reaction to occur. We demonstrate the benefits of analyzing these enzymes in their reaction buffer, uncovering trends that were not observable in the typical phosphate storage buffer. This work provides a framework for analyzing the stability of many other enzymes whose reaction media is not suitable for traditional techniques. We introduce several strategies for measuring the melting temperature, oligomeric state, and activity of these enzymes in their reaction media. Further, we have identified opportunities for integration of computational tools into this workflow to engineer enzymes more effectively for solvent tolerance and improved stability.