Structure最新文献

Designing antibodies is complex and resource intensive. While deep learning and generative approaches have shown promise in the design of protein binders, achieving high affinity and stability remains challenging. We introduce EvolveX, a structure-based antibody design pipeline leveraging the empirical force field FoldX to design complementarity-determining regions (CDRs) of single-domain antibodies (VHHs). We demonstrate the ability of EvolveX to redesign a VHH targeting mouse Vsig4 (mVsig4) to address two challenges: enhancing stability and affinity for mVsig4 and redesigning it for high affinity to the human ortholog. Notably, EvolveX improved the binding affinity of VHHs to human Vsig4 by over 1,000-fold. Structural analyses by X-ray crystallography confirmed design accuracy. Next-generation sequencing (NGS) analysis further demonstrated the efficiency of FoldX-based design pipeline. Collectively, our study highlights EvolveX's potential to overcome current limitations in antibody design, offering a powerful tool for the development of therapeutics with enhanced specificity, stability, and efficacy.

In this meet-the-author Q&A, Structure's editor-in-chief, Karin Kühnel, speaks to Cathleen Zeymer from the Technical University of Munich about her research group's recent Structure paper entitled "Modular protein scaffold architecture and AI-guided sequence optimization facilitate de novo metalloenzyme engineering" and her work and career.

DNA mismatch repair is an evolutionarily conserved repair pathway that corrects replication errors, thereby preventing genome instability. Two evolutionarily conserved proteins, MutS and MutL, recognize the mismatch and mark the newly synthesized strand for repair. Previous studies have shown how bacterial MutS homodimers function asymmetrically to recognize mismatches and recruit MutL. However, whether MutL homodimers also function asymmetrically to coordinate binding to MutS and activation of their nuclease activity remains unclear. Here, we characterize the ATPase domain of Bacillus subtilis MutL, a MutL protein with endonuclease activity, and delineate the differences with Escherichia coli MutL, a homolog without endonuclease activity. We find that B. subtilis MutL has low affinity for ATP and samples a repertoire of conformations that resemble those observed in eukaryotic MutL paralogs, indicating a relationship between ATP-induced dimer compaction and nuclease activity.

Dengue virus infection remains a public health threat. Dengue NS2B-NS3 proteins are prime antiviral drug targets, highly dynamic, and adopt different structural conformations. We combine cross-linking mass spectrometry (XL-MS), molecular dynamics (MD) simulations, and biochemical assays to identify NS2B-NS3 full length interactions. Using cross-linkers of different lengths as molecular rulers, we identified NS2B S48 as a key interacting residue with NS3 by XL-MS. Structural modeling with MD simulations revealed a novel compact conformation of the NS2B-NS3 complex. Mutation of NS2B S48 to alanine or lysine greatly reduced protease activity and disrupted the binding pocket in MD simulations with a loss of NS2B-NS3 interactions. Additionally, NS2B-NS3 cross-links were found to be conserved across all four dengue serotypes. Our interdisciplinary approach reveals a new key interacting residue and a compact conformation that are structurally and functionally important for the dynamic NS2B-NS3 complex. These results can help guide drug development against dengue.

The increasing global incidence rate of nontuberculous mycobacteria pulmonary infections is an emerging public health crisis, with Mycobacterium abscessus (Mab) being one of the most virulent and treatment-refractory of these pathogens. Mab exhibits extensive intrinsic and acquired drug resistance mechanisms that neutralize most antimicrobials against this pathogen, causing a clinical conundrum. As Mab relies on oxidative phosphorylation as its main energy source, its essential F-ATP synthase is a promising drug target but remains poorly understood due to a lack of host expression systems. Here, we present the expression, isolation, and structural characterization of Mab's F-ATP synthase. Cryo-EM reveals three nucleotide-driven rotational states at atomic resolution, highlighting key catalytic centers, a mycobacteria-specific α-subunit extension involved in the inhibition of ATP hydrolysis, energy transmission via the γε-stalk, and mechanochemical coupling by the δ-subunit. The structural blueprint allows precise target engagement and optimization of hits-to-leads and existing anti-Mab inhibitors targeting the engine.

The neuronal α7 nicotinic acetylcholine receptor (α7-nAChR) and muscle-type nicotinic acetylcholine receptor (mt-nAChR) are pivotal in synaptic signaling within the brain and the neuromuscular junction respectively. Additionally, they are both targets of a wide range of drugs and toxins. Here, we utilize cryo-EM to delineate structures of these nAChRs in complex with the conotoxins ImI and ImII from Conus imperialis. Despite nominal sequence differences, ImI and ImII exhibit discrete binding preferences and adopt drastically different conformational states upon binding. ImI engages the orthosteric sites of α7-nAChR, while ImII forms distinct pore-bound complexes with both α7-nAChR and mt-nAChR. Strikingly, ImII adopts a compact globular conformation that binds as a monomer to the α7-nAChR pore and as an oblate dimer to the mt-nAChR pore. These structures advance our understanding of nAChR-ligand interactions and the subtle sequence variations that result in dramatically altered functional outcomes in small peptide toxins.