Structure最新文献

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.

Tubulin family proteins play central roles in the organization and dynamics of cytoskeletal systems across the Tree of Life. In one family of bacteriophages (phages), tubulin-like proteins called PhuZ (Phage tubulin/FtsZ) form dynamic filaments that position and rotate an intracellular compartment, the "phage nucleus," in which the phage genome is replicated. PhuZ filaments also mediate trafficking of nascent capsids from the cell periphery to the phage nucleus for genome packaging. PhuZ from the Pseudomonas-infecting phages 201Phi2-1 and PhiKZ form assemblies with three protofilaments. Here, we determine a 2.8 Å resolution structure of PhuZ from the E. coli-infecting phage Goslar, which forms an elaborate "cytoskeletal vortex" in infected cells. We find that in vitro-assembled Goslar PhuZ forms rigid tubes with nine nearly-straight protofilaments. The lateral interactions mediating this assembly are fundamentally different from eukaryotic tubulin, leading to a distinctive overall architecture for Goslar PhuZ filaments.

The non-receptor tyrosine phosphatase SHP2 (SH2 domain-containing protein tyrosine phosphatase 2) (PTPN11) is a regulator of diverse cellular functions including mitogenic activation and cell migration. It comprises two tandem Src-homology 2 (SH2) domains followed by the catalytic domain and is autoinhibited by the N-terminal SH2 domain blocking access to the active site. Mutations influencing auto-inhibition have been implicated in cancer and other diseases, and allosteric inhibitors have been developed that stabilize the inactive state. Here, we show that the intrinsically disordered bis-phosphorylated SHP2-activating peptide pY⁶²⁷pY⁶⁵⁹-Gab1 binds to both SH2 domains, undergoing partial ordering in the process. In addition to eliciting changes in SH2 domain dynamics, the peptide reorganizes their relative orientations to generate a new SH2-SH2 interface. Our data suggest an active conformation for SHP2 that is also applicable to the hematopoietic cell-specific SHP1 (PTPN6), shedding light on the activation mechanism of both enzymes and paving the way for the development of novel compounds to modulate SHP2 activity.