Direct experimental observation of blue-light-induced conformational change and intermolecular interactions of cryptochrome.

Cryptochromes are blue light receptors that mediate circadian rhythm and magnetic sensing in various organisms. A typical cryptochrome consists of a conserved photolyase homology region domain and a varying carboxyl-terminal extension across species. The structure of the flexible carboxyl-terminal extension and how carboxyl-terminal extension participates in cryptochrome's signaling function remain mostly unknown. In this study, we uncover the potential missing link between carboxyl-terminal extension conformational changes and downstream signaling functions. Specifically, we discover that the blue-light induced opening of carboxyl-terminal extension in C. reinhardtii animal-like cryptochrome can structurally facilitate its interaction with Rhythm Of Chloroplast 15, a circadian-clock-related protein. Our finding is made possible by two technical advances. Using single-molecule Förster resonance energy transfer technique, we directly observe the displacement of carboxyl-terminal extension by about 15 Å upon blue light excitation. Combining structure prediction and solution X-ray scattering methods, we propose plausible structures of full-length cryptochrome under dark and lit conditions. The structures provide molecular basis for light active conformational changes of cryptochrome and downstream regulatory functions.

Computational insights into differential interaction of mammalian angiotensin-converting enzyme 2 with the SARS-CoV-2 spike receptor binding domain.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Angiotensin-converting enzyme 2 (ACE2) has been identified as the host cell receptor that binds to the receptor-binding domain (RBD) of the SARS-COV-2 spike protein and mediates cell entry. Because the ACE2 proteins are widely available in mammals, it is important to investigate the interactions between the RBD and the ACE2 of other mammals. Here we analyzed the sequences of ACE2 proteins from 16 mammals, predicted the structures of ACE2-RBD complexes by homology modeling, and refined the complexes using molecular dynamics simulation. Analyses on sequence, structure, and dynamics synergistically provide valuable insights into the interactions between ACE2 and RBD. The analysis outcomes suggest that the ACE2 of bovine, cat, and panda form strong binding interactions with RBD, while in the cases of rat, least horseshoe bat, horse, pig, mouse, and civet, the ACE2 proteins interact weakly with RBD.

Structure-Based Functional Modification Study of a Cyanobacterial Chloride Pump for Transporting Multiple Anions.

Understanding the structure and functional mechanisms of cyanobacterial halorhodopsin has become increasingly important, given the report that Synechocystis halorhodopsin (SyHR), a homolog of the cyanobacterial halorhodopsin from Mastigocladopsis repens (MrHR), can take up divalent ions, such as SO42-, as well as chloride ions. Here, the crystal structure of MrHR, containing a unique "TSD" chloride ion conduction motif, was determined as a homotrimer at a resolution of 1.9 Å. The detailed structure of MrHR revealed a unique trimeric topology of the light-driven chloride pump, with peculiar coordination of two water molecules and hydrogen-mediated bonds near the TSD motif, as well as a short B-C loop. Structural and functional analyses of MrHR revealed key residues responsible for the anion selectivity of cyanobacterial halorhodopsin and the involvement of two chloride ion-binding sites in the ion conduction pathway. Alanine mutant of Asn63, Pro118, and Glu182 locating in the anion inlet induce multifunctional uptake of chloride, nitrate, and sulfate ions. Moreover, the structure of N63A/P118A provides information on how SyHR promotes divalent ion transport. Our findings significantly advance the structural understanding of microbial rhodopsins with different motifs. They also provide insight into the general structural framework underlying the molecular mechanisms of the cyanobacterial chloride pump containing SyHR, the only molecule known to transport both sulfate and chloride ions.

Computer aided protein engineering to enhance the thermo-stability of CXCR1- T4 lysozyme complex.

CXCR1, a member in G-protein coupled receptor (GPCR) family, binds to chemokine interleukin-8 (IL-8) specifically and transduces signals to mediate immune and inflammatory responses. Despite the importance of CXCR1, high-resolution structure determination is hindered by the challenges in crystallization. It has been shown that properly designed mutants with enhanced thermostability, together with fusion partner proteins, can be useful to form crystals for GPCR proteins. In this study, in silico protein design was carried out by using homology modeling and molecular dynamics simulations. To validate the computational modeling results, the thermostability of several mutants and the wild type were measured experimentally. Both computational results and experimental data suggest that the mutant L126W has a significant improvement in the thermostability. This study demonstrated that in silico design can guide protein engineering and potentially facilitate protein crystallography research.

Identification of Drug Binding Sites and Action Mechanisms with Molecular Dynamics Simulations.

Identifying drug binding sites and elucidating drug action mechanisms are important components in a drug discovery process. In this review, we briefly compared three different approaches (sequence- based methods, structure-based methods and probe-based molecular dynamics (MD) methods) to identifying drug binding sites, and concluded that probe-based MD methods are much more advantageous in dealing with flexible target macromolecules and digging out druggable macromolecule conformations for subsequent drug screening. The applications of MD simulation to studying drug-target interactions were demonstrated with different types of target molecules, including lipid membrane, protein and DNA. The results indicate that MD simulations with enhanced sampling methods provide a powerful tool to determine free energy profiles/surfaces and identify important intermediate states, which are essential for the elucidation of drug action mechanisms. The future development of methods in MD simulations will benefit and speed up the drug discovery processes.