Biochemistry Biochemistry最新文献

The thiol group of the Cys side chain is known to participate in hydrogen bonds as an acceptor or donor. Similarly, the backbone nitrogens in proteins are involved in forming hydrogen bonds as donors that provide stability to protein secondary structures. In this study, we have identified more than 400 examples of self-contacting and inter-residue contacts from nearly 6000 high-resolution protein crystal structures in which the S–H group of the Cys side chain and the backbone nitrogen satisfy the geometric criteria to form hydrogen bonds. Very few studies have investigated the role of backbone nitrogen as a hydrogen-bond acceptor. Relative energy profiles calculated by varying the Cys χ¹ side chain dihedral angle of self-contacting Cys residues revealed that the energy difference between crystal structure and minimum energy conformations is between 0–3 kcal/mol. Quantum chemical calculations using DFT and MP2 theories indicated that the interaction energies of model systems with S–H···N self-contacts were only marginally favorable. However, the model systems representing S–H···N inter-residue contacts showed reasonably stable interaction. Natural bond orbital (NBO) analysis and NCIPLOT studies do not exhibit any hydrogen-bond interaction between the S–H donor and acceptor backbone nitrogen. The favorable interaction energies may be due to electrostatic and dispersion interactions. We found that the interactions due to S–H···N inter-residue contacts stabilize two secondary structural elements, and a large number of them occur between two β-strands. The structural role of S–H···N interactions can be further investigated by mutation studies of specific Cys residues involved in S–H···N contacts.

Conformational dynamics is a fundamental aspect of enzymatic catalysis that, for example, can be linked to ligand binding and release, assembly of the active site, and the catalytic mechanism. The essential and metabolic enzyme adenylate kinase (AK) undergoes large-scale conformational changes in response to binding of its substrates ATP and AMP. As such, it has been intensely studied in search of linkages between dynamics and catalysis. For a complex conformational change to occur in a protein, whether it is of an induced fit or conformational selection nature, changes at several hinges are often required. Here, based on a comparative structure–function analysis of AK enzymes from E. coli and the archaea Odinarchaeota and from human AK1, we found that conformational changes in the enzymes are to a varying degree linked to bending, fraying, or unfolding/folding events of the termini of α-helices observed in various structural hot spots of the enzymes. The findings contribute with a mechanistic angle to how enzymatic dynamics and catalysis relate to the plasticity of the termini of α-helices.

Protein homeostasis is tightly controlled by the coordinated actions of E3 ubiquitin ligases and deubiquitinases (DUBs). We previously identified Spindlin-4 (SPIN4), a histone H3K4me3 reader, as a degradation substrate of DCAF16. In this study, we confirmed this degradation pathway using an E3 ligase-focused CRISPR-Cas9 knockout screen. Furthermore, through a DUB-focused CRISPR-Cas9 knockout screen and biochemical analyses, we demonstrated that the deubiquitinase BAP1 interacts with and stabilizes SPIN4 via its deubiquitination activity. Inhibition or loss of BAP1 reduces SPIN4 levels, highlighting its critical role in maintaining SPIN4 homeostasis. Proteomics and interactome analyses further support this regulatory axis. These findings reveal a dynamic balance controlling SPIN4 stability, with potential implications for epigenetic regulation and disease processes.

In this study, we present an atomic-level structural investigation of the magainin 2 antimicrobial peptide reconstituted in extended lipid bilayers that closely mimic the composition of bacterial membranes. Using state-of-the-art solid-state NMR spectroscopy, we show that within liquid-crystalline membranes the peptide exhibits site-specific motional regimes, which correlate with its amphipathic character. Peptide-lipid interactions are identified at the polar headgroup region consistent with an in-plane topology also observed by oriented ¹⁵N solid-state NMR spectroscopy. While ¹³C chemical shift analysis reveals α-helical conformations, the NMR line shapes indicate pronounced conformational heterogeneity, which can be explained by the existence of higher order arrangements along the membrane surface. A reduced degree of helicity is observed when the membrane is in the gel phase suggesting more superficial interactions of magainin 2. Notably, our NMR data show that membrane-associated magainin 2 can evolve into amyloid-like β-sheet structures, forming large peptide-lipid aggregates. This behavior occurs only in bacterial and not in mammalian membrane models, paving the way for a new understanding of the role of these supramolecular assemblies in host defense mechanisms, and highlighting a potential relationship between antimicrobial peptides and functional amyloid structures.

Motile cilia and flagella are complex microtubule-based organelles essential for cell motility, fluid flow, and sensory functions. At the core of these organelles lie doublet microtubules (DMTs), specialized structures consisting of an A-tubule and a B-tubule that serve as the scaffold for axonemal complexes. Recent advances have uncovered the intricate architecture of DMTs, highlighting inner and outer junction proteins, luminal scaffolds, and interdoublet linkers critical for structural stability and function. Studies in model organisms such as Chlamydomonas, Tetrahymena, and Caenorhabditis elegans have identified conserved regulators orchestrating DMT assembly. Parallel insights from human genetics reveal that mutations in DMT-associated proteins underlie a subset of cases of primary ciliary dyskinesia and other ciliopathies. This review synthesizes current understanding of DMT biogenesis from molecular, structural, and disease perspectives, illuminating how coordinated assembly ensures ciliary function and how its disruption leads to human disease.

Chemigenetic indicators combined with advanced organic fluorophores have become increasingly popular in biosensor development due to their integration of protein biocompatibility and the superior optical properties of dyes. However, despite the development of various biosensors based on these systems, the underlying activation mechanisms remain obscure. Here, we report the first crystal structure of the recently developed calcium indicator WHaloCaMP1a in complex with the rhodamine-based fluorophore BD566_HTL. Integrating structural analysis with molecular dynamics simulations, we identify a potential activation mechanism of WHaloCaMP1a and reveal novel interactions between the sensor and BD566. These findings provide new insights for the rational design of biosensors and the study of protein–dye coevolution.

Xeruborbactam is a boronic acid-based transition-state analogue that has exhibited great potential as a clinically relevant inhibitor of carbapenemase enzymes, including class A carbapenemases. In this work, we have investigated the mechanism of inhibition of xeruborbactam against SME-1 carbapenemase using kinetic, structural, and thermodynamic approaches. With a K_i(app) of 4 nM, xeruborbactam shows more potent inhibitory activity than any other beta-lactamase inhibitor available until now. Structural data from crystal complexes revealed that xeruborbactam covalently engages Ser70 at the active site and forms stabilizing interactions; in particular, the cyclopropyl group forms hydrophobic interactions with His105, further stabilizing the adduct, which correlates with a high rate of borylation and minimal deborylation. We investigated xeruborbactam with its 2R,3S-cyclopropyl isomer to grasp the influence of the stereochemistry of the cyclopropyl ring. Although both inhibitors bind covalently to Ser70 in SME-1, the 2R,3S-isomer adopts a different conformation of the cyclopropyl ring, which makes the C3 carbon much farther from His105, Asn132, and Lys73, thereby decreasing the binding affinity and K_i(app) of the isomer. Furthermore, the fluorine-12 atom takes different conformations in the two structures, changing the terrain of interaction with the protein. Consistent with its lowered inhibition efficiency, the 2R,3S-isomer shows a lower borylation rate and weaker enzyme–inhibitor binding. In the molecular dynamics, xeruborbactam stabilized SME-1 more than its isomer, which is consistent with our experimental findings. These results together show the strong inhibitory profile of xeruborbactam and highlight the importance of stereochemistry in the design of next-generation β-lactamase inhibitors and diagnostics for AMR.

The small heat shock proteins (sHsps) are a key class of molecular chaperones that can inhibit protein aggregation and enhance protein recovery from aggregates. However, the mechanisms sHsps employ to carry out these roles are not well understood, in part because the highly heterogeneous and dynamic particles they form with aggregating proteins are difficult to study with traditional biophysical tools. Here we have applied a novel single particle fluorescence technique known as Burst Analysis Spectroscopy (BAS) to the study of the Escherichia coli sHsps IbpA and IbpB (IbpAB). We show that in the presence of IbpAB, two different model proteins converge toward similar, limited aggregate particle size distributions. Additionally, while IbpAB dramatically accelerates the disassembly of protein aggregates by the bacterial KJEB bichaperone disaggregase, this enhancement does not appear to be strongly influenced by aggregate particle size. Rather, it is the ability of IbpAB to alter aggregate structure during particle formation that appears to be essential for stimulated disassembly. These observations support a model of aggregate recognition by IbpAB that is not only highly adaptable but capable of shaping aggregate particles into a specialized range of physical properties that are necessary for efficient protein disaggregation.