Structure of tetrameric forms of the serotonin-gated 5-HT3A receptor ion channel.

Multimeric membrane proteins are produced in the endoplasmic reticulum and transported to their target membranes which, for ion channels, is typically the plasma membrane. Despite the availability of many fully assembled channel structures, our understanding of assembly intermediates, multimer assembly mechanisms, and potential functions of non-standard assemblies is limited. We demonstrate that the pentameric ligand-gated serotonin 5-HT3A receptor (5-HT3AR) can assemble to tetrameric forms and report the structures of the tetramers in plasma membranes of cell-derived microvesicles and in membrane memetics using cryo-electron microscopy and tomography. The tetrameric structures have near-symmetric transmembrane domains, and asymmetric extracellular domains, and can bind serotonin molecules. Computer simulations, based on our cryo-EM structures, were used to decipher the assembly pathway of pentameric 5-HT3R and suggest a potential functional role for the tetrameric receptors.

Clinical and pathological features of 52 patients with glomerulonephritis with dominant C3. / C3沉积为主的肾小球肾炎52ä¾‹ä¸´åºŠä¸Žç— ç†ç‰¹å¾.

OBJECTIVES: With the in-depth study of complement dysregulation, glomerulonephritis with dominant C3 has received increasing attention, with a variety of pathologic types and large differences in symptoms and prognosis between pathologic types. This study analyzes the clinical, pathological, and prognostic characteristics of different pathological types of glomerulonephritis with dominant C3, aiming to avoid misdiagnosis and missed diagnoses. METHODS: The clinical, pathological, and follow-up data of 52 patients diagnosed as glomerulonephritis with dominant C3 by renal biopsy from June 2013 to October 2022 were retrospectively analyzed. According to the clinical feature and results of pathology, 15 patients with post-infectious glomerulonephritis (PIGN) and 37 patients with of non-infectious glomerulonephritis (N-PIGN) were classified. N-PIGN subgroup analysis was performed, and 16 patients were assigned into a C3-alone-deposition group and 21 in a C3-dominant-deposition group, or 27 in a C3 glomerulopathy (C3G) group and 10 in a non-C3 nephropathy (N-C3G) group. RESULTS: The PIGN group had lower creatinine values (84.60 µmol/L vs179.62 µmol/L, P=0.001), lower complement C3 values (0.36 g/L vs0.74 g/L, P<0.001) at biopsy, and less severe pathological chronic lesions compared with the N-PIGN group. In the N-PIGN subgroup analysis, the C3-dominant-deposition group had higher creatinine values (235.30 µmol/L vs106.70 µmol/L, P=0.004) and higher 24-hour urine protein values (4 025.62 mg vs1 981.11 mg, P=0.037) than the C3-alone-deposition group. The prognosis of kidney in the PIGN group (P=0.049), the C3-alone-deposition group (P=0.017), and the C3G group (P=0.018) was better than that in the N-PIGN group, the C3-dominant-deposition group, and the N-C3G group, respectively. CONCLUSIONS: Glomerulonephritis with dominant C3 covers a variety of pathological types, and PIGN needs to be excluded before diagnosing C3G because of considerable overlap with atypical PIGN and C3G; in addition, the deposition of C1q complement under fluorescence microscope may indicate poor renal prognosis, and relevant diagnosis, treatment, and follow-up should be strengthened.

S-nitrosylation of a receptor-like cytoplasmic kinase regulates plant immunity.

Perception of pathogen/microbial-associated molecular patterns (P/MAMPs) by plant cell surface receptors leads to a sustained burst of reactive oxygen species (ROS), a key feature of P/MAMP-triggered immunity (PTI). Here we report that P/MAMP recognition leads to a rapid nitrosative burst, initiating the accumulation of nitric oxide (NO), subsequently leading to S-nitrosylation of the receptor-like cytoplasmic kinase (RLCK), botrytis-induced kinase 1 (BIK1), at Cys80. This redox-based, posttranslational modification, promotes the phosphorylation of BIK1, subsequently resulting in BIK1 activation and stabilization. Further, BIK1 S-nitrosylation increases its physical interaction with RBOHD, the source of the apoplastic oxidative burst, promoting ROS formation. Our data identify mechanistic links between rapid NO accumulation and the expression of PTI, providing insights into plant immunity.

Machine Learning Deciphered Molecular Mechanistics with Accurate Kinetic and Thermodynamic Prediction.

Time-lagged independent component analysis (tICA) and the Markov state model (MSM) have been extensively employed for extracting conformational dynamics and kinetic community networks from unbiased trajectory ensembles. However, these techniques may not be the optimal choice for elucidating transition mechanisms within low-dimensional representations, especially for intricate biosystems. Unraveling the association mechanism in such complex systems always necessitates permutations of several essential independent components or collective variables, a process that is inherently obscure and may require empirical knowledge for selection. To address these challenges, we have implemented an integrated unsupervised dimension reduction model: uniform manifold approximation and projection (UMAP) with hierarchy density-based spatial clustering of applications with noise (HDBSCAN). This approach effectively generates low-dimensional configurational embeddings. The hierarchical application of this architecture, in conjunction with MSM, reveals global kinetic connectivity while identifying local conformational states. Consequently, our methodology establishes a multiscale mechanistic elucidation framework. Leveraging the benefits of the uniform sample distribution and a denoising approach, our model demonstrates robustness in preserving global and local data structures compared to traditional dimension reduction methods in the field of MD analysis area. The interpretability of hyperparameter selection and compatibility with downstream tasks are cross-validated across various simulation data sets, utilizing both computational evaluation metrics and experimental kinetic observables. Furthermore, the predicted Mcl1-BH3 association kinetics (0.76 s-1) is in close agreement with surface plasmon resonance experiments (0.12 s-1), affirming the plausibility of the identified pathway composed of representative conformations. We anticipate that the devised workflow will serve as a foundational framework for studying recognition patterns in complex biological systems. Its contributions extend to the exploration of protein functional dynamics and rational drug design, offering a potent avenue for advancing research in these domains.

High-expression of FABP4 in Tubules is a Risk Factor for Poor Prognosis in DKD Patients.

BACKGROUND: Lipid metabolism imbalance is involved in the mechanism of renal tubular injury in diabetic kidney disease (DKD). Fatty acid binding protein 4 (FABP4) has been reported to participate in cellular lipid toxicity. However, the expression of FABP4 in renal tissues of DKD and its correlation with clinical/ pathological parameters and prognosis have not been studied. METHODS: A retrospective cohort study was conducted in 108 hospitalized Type 2 diabetes (T2D) patients with renal injury, including 70 with DKD and 38 with NDKD (non-DKD). Clinical features, pathological findings, and follow-up parameters were collected. Serum and urine FABP4 were detected by ELISA. An immunohistochemistry stain was used to determine FABP4 in renal tubulointerstitium. A double immunofluorescence stain was employed to assess FABP4- and CD68-positive macrophages. Correlation analysis, logistic regression models, receiver operating characteristic (ROC), and Kaplan-Meier survival curve were performed for statistical analysis. RESULTS: DKD patients had increased expression of FABP4 and ectopic fat deposition in tubules. As shown by correlation analyses, FABP4 expression in renal tubules was positively correlated with UNAG (r=0.589, p=0.044) and ESRD (r=0.740, p=0.004). Multivariate regression analysis revealed that UNAG level was correlated with FABP4 expression level above median value (odds ratio:1.154, 95% confidence interval:1.009-1.321, p=0.037). High-expression of FABP4 in renal tubules of DKD was at an increased risk of ESRD. Increased FABP4 expression in inflammatory cells was also associated with ESRD in DKD. CONCLUSION: High-expression of FABP4 is involved in the pathogenesis of renal tubular lipid injury and is a risk factor for poor prognosis in DKD patients.

Computational drug development for membrane protein targets.

The application of computational biology in drug development for membrane protein targets has experienced a boost from recent developments in deep learning-driven structure prediction, increased speed and resolution of structure elucidation, machine learning structure-based design and the evaluation of big data. Recent protein structure predictions based on machine learning tools have delivered surprisingly reliable results for water-soluble and membrane proteins but have limitations for development of drugs that target membrane proteins. Structural transitions of membrane proteins have a central role during transmembrane signaling and are often influenced by therapeutic compounds. Resolving the structural and functional basis of dynamic transmembrane signaling networks, especially within the native membrane or cellular environment, remains a central challenge for drug development. Tackling this challenge will require an interplay between experimental and computational tools, such as super-resolution optical microscopy for quantification of the molecular interactions of cellular signaling networks and their modulation by potential drugs, cryo-electron microscopy for determination of the structural transitions of proteins in native cell membranes and entire cells, and computational tools for data analysis and prediction of the structure and function of cellular signaling networks, as well as generation of promising drug candidates.

The structure and function of olfactory receptors.

Olfactory receptors (ORs) form the most important chemosensory receptor family responsible for our sense of smell in the nasal olfactory epithelium. This receptor family belongs to the class A G protein-coupled receptors (GPCRs). Recent research has indicated that ORs are involved in many nonolfactory physiological processes in extranasal tissue, such as the brain, pancreas, and testes, and implies the possible role of their dysregulation in various diseases. The recently released structures of OR51E2 and consensus OR52 have also unveiled the uniqueness of ORs from other class A GPCR members. In this review, we discuss these recent developments and computational modeling efforts toward understanding the structural properties of unresolved ORs, which could guide potential future OR-targeted drug discovery.

Machine learning for data-driven design of high-safety lithium metal anode.

Here, we present a protocol for developing an inorganic-organic hybrid interphase layer using the self-assembled monolayers technique to enhance the surface of the lithium metal anode. We describe steps for extracting organic molecules from open-sourced databases and calculating their microscopic properties. We then detail procedures for developing a machine learning model for predicting the ionic diffusion barrier and preparing the inputs for prediction. This protocol enables a cost-effective workflow to identify promising self-assembled monolayers with exceptional performance. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2023).1.

CD4+ T-Cell Legumain Deficiency Attenuates Hypertensive Damage via Preservation of TRAF6.

BACKGROUND: T cells are central to the immune responses contributing to hypertension. LGMN (legumain) is highly expressed in T cells; however, its role in the pathogenesis of hypertension remains unclear. METHODS: Peripheral blood samples were collected from patients with hypertension, and cluster of differentiation (CD)4+ T cells were sorted for gene expression and Western blotting analysis. TLGMNKO (T cell-specific LGMN-knockout) mice (Lgmnf/f/CD4Cre), regulatory T cell (Treg)-specific LGMN-knockout mice (Lgmnf/f/Foxp3YFP Cre), and RR-11a (LGMN inhibitor)-treated C57BL/6 mice were infused with Ang II (angiotensin II) or deoxycorticosterone acetate/salt to establish hypertensive animal models. Flow cytometry, 4-dimensional label-free proteomics, coimmunoprecipitation, Treg suppression, and in vivo Treg depletion or adoptive transfer were used to delineate the functional importance of T-cell LGMN in hypertension development. RESULTS: LGMN mRNA expression was increased in CD4+ T cells isolated from hypertensive patients and mice, was positively correlated with both systolic and diastolic blood pressure, and was negatively correlated with serum IL (interleukin)-10 levels. TLGMNKO mice exhibited reduced Ang II-induced or deoxycorticosterone acetate/salt-induced hypertension and target organ damage relative to wild-type (WT) mice. Genetic and pharmacological inhibition of LGMN blocked Ang II-induced or deoxycorticosterone acetate/salt-induced immunoinhibitory Treg reduction in the kidneys and blood. Anti-CD25 antibody depletion of Tregs abolished the protective effects against Ang II-induced hypertension in TLGMNKO mice, and LGMN deletion in Tregs prevented Ang II-induced hypertension in mice. Mechanistically, endogenous LGMN impaired Treg differentiation and function by directly interacting with and facilitating the degradation of TRAF6 (tumor necrosis factor receptor-associated factor 6) via chaperone-mediated autophagy, thereby inhibiting NF-κB (nuclear factor kappa B) activation. Adoptive transfer of LGMN-deficient Tregs reversed Ang II-induced hypertension, whereas depletion of TRAF6 in LGMN-deficient Tregs blocked the protective effects. CONCLUSIONS: LGMN deficiency in T cells prevents hypertension and its complications by promoting Treg differentiation and function. Specifically targeting LGMN in Tregs may be an innovative approach for hypertension treatment.

Changes in spectrum of biopsy-proven kidney diseases within decade: an analysis based on 10 199 cases from South China.

PURPOSE: To assess the regional epidemiological trends of kidney diseases over time in the South China using renal biopsy-proven cases. METHODS: This retrospective observational cohort study was conducted at the Institute of Nephrology, Second Xiangya Hospital of Central South University, and encompasses all patients diagnosed with kidney disease via biopsy from 2012 to 2021. RESULTS: The study sample consisted of 10 199 native kidneys, with a male-to-female ratio of 0.91:1 and an average age of 38.74 (±14.53) years. Primary glomerular nephropathy, systemic glomerular nephropathy (SGN), tubulointerstitial disease, and hereditary renal diseases accounted for 66.92 (6825)%, 24.49 (2498)%, 8.06 (822)%, and 0.53 (54)%, respectively. The leading pathologies of primary glomerular nephropathy remained the IgA nephropathy. The frequencies of IgA nephropathy and membranous nephropathy increased significantly, whereas the frequencies of minimal change disease and focal segmental glomerulosclerosis decreased (P < .001) between 2017 and 2021 than in the years 2012 and 2016. An earlier onset of membranous nephropathy was observed in the age group of 45-59 years compared to previous studies. The leading pathologies of SGN were found to be lupus nephritis (758 cases, 30.45%) and hypertension nephropathy (527 cases, 21.17%). The frequencies of hypertension nephropathy and diabetic nephropathy increased between 2017 and 2021 compared to 2012 and 2016 (P < .001), gradually becoming the leading pathological types of SGN. In elderly patients diagnosed with nephrotic syndrome, the frequencies of amyloidosis significantly increased (P < .01). CONCLUSION: Our study may provide insights for kidney disease prevention and public health strategies. What is already known on this topic The pathological spectrum of kidney diseases has undergone significant transformations in the past decade, driven by the escalating incidence of chronic diseases. Although there are studies exploring the renal biopsy findings from various regions in China which present both similarities and differences in epidemiology, few large-scale reports from the South China in recent decades were published. What this study adds Our findings reveal the following key observations: (i) increased proportion of middle-aged patients leading to the increasing average age at the time of biopsy；(ii) the frequencies of IgA nephropathy and membranous nephropathy (MN) increased significantly, whereas the frequencies of minimal change disease and focal segmental glomerulosclerosis decreased (P < .001) between 2017 and 2021 than in the years 2012 and 2016; (iii) earlier onset of MN in the age group of 45-59 years old was found in our study; and (iv) a higher frequency of hypertension nephropathy and DN presented over time, and frequency of amyloidosis increased in elderly patients diagnosed with NS. How this study might affect research, practice, or policy This single-center yet a large-scale study of the kidney disease spectrum in South China may provide a reference point for the diagnosis, treatment, and prevention of chronic kidney disease.

Inhibitor screening for volume-sensitive LRRC8A chloride channel.

Leucine-rich repeat-containing protein 8 A (LRRC8A) protein is a critical member of volume-regulated anion channels. It plays a critical roles in the regulation of cellular volume and involves in the development of diseases like osteoarthritis. Screening of lead compounds to modulate its function may provide potential therapeutics of related diseases. Here, we employ virtual screening techniques and molecular dynamics (MD) simulation to screen potential inhibitors against LRRC8A. LRRC8A was regarded as the drug target to investigate potential compounds from the ZINC15 database via molecular docking. The final compound was selected among the top 10 Autodock Vina score (-8.8 Kcal/mol) with the ZINC ID ZINC000018195627 after druggability prediction. The docked complex from the virtual screening was subjected to MD simulation to analyze the stability of the LRRC8A protein-ligand complex, with parameters including root mean square deviation, root mean square fluctuation and radius of gyration. Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method was further employed to predict the binding free energies from MD simulation trajectory. Our study provides insightful analysis for the potential compound to modulate LRRC8A and lay the foundation of therapeutics development against osteoarthritis.Communicated by Ramaswamy H. Sarma.

Clinical and pathological features of omicron variant of SARS-CoV-2-associated kidney injury.

Kidney injury is common in patients with Coronavirus Disease-19 (COVID-19), which is related to poor prognosis. We aim to summarize the clinical features, athological types, and prognosis of COVID-19 associated kidney injury caused by the Omicron strain. In this study, 46 patients with Omicron-associated kidney injury were included, 38 of whom performed renal biopsy. Patients were divided into two groups: group A for patients with onset of kidney injury after SARS-CoV-2 infection; group B for patients with pre-existing kidney disease who experienced aggravation of renal insufficiency after SARS-CoV-2 infection. The clinical, pathological, and prognostic characteristics of the patients were observed. Acute kidney injury (AKI) (35%) was the most common clinical manifestation in group A. Patients in group B mainly presented with chronic kidney disease (CKD) (55%) and nephrotic syndrome (NS) (40%). The pathological type was mainly IgA nephropathy (IgAN) (39% in group A and 45% in group B). Among all of them, one case presenting with thrombotic microangiopathy had worse kidney function at biopsy time. Mean serum C3 levels were 1.2 ± 0.5 and 1.0 ± 0.2 g/L in group A and group B, respectively. In renal tissues, C3 deposits were observed in 71.1% of patients. 11.8% (n = 2) patients experienced deterioration of renal function after treatment, but no patients developed to end-stage renal disease. In our single-center study in China, the main clinical manifestations were AKI, CKD, and NS, while the main pathological type was IgAN. Compared with previous strains of SARS-CoV-2, patients with the Omicron infection had a favorable prognosis.

DsbA-L interacting with catalase in peroxisome improves tubular oxidative damage in diabetic nephropathy.

Peroxisomes are metabolically active organelles that are known for exerting oxidative metabolism, but the precise mechanism remains unclear in diabetic nephropathy (DN). Here, we used proteomics to uncover a correlation between the antioxidant protein disulfide-bond A oxidoreductase-like protein (DsbA-L) and peroxisomal function. In vivo, renal tubular injury, oxidative stress, and cell apoptosis in high-fat diet plus streptozotocin (STZ)-induced diabetic mice were significantly increased, and these changes were accompanied by a "ghost" peroxisomal phenotype, which was further aggravated in DsbA-L-deficient diabetic mice. In vitro, the overexpression of DsbA-L in peroxisomes could improve peroxisomal phenotype and function, reduce oxidative stress and cell apoptosis induced by high glucose (HG, 30 mM) and palmitic acid (PA, 250 µM), but this effect was reversed by 3-Amino-1,2,4-triazole (3-AT, a catalase inhibitor). Mechanistically, DsbA-L regulated the activity of catalase by binding to it, thereby reducing peroxisomal leakage and proteasomal degradation of peroxisomal matrix proteins induced by HG and PA. Additionally, the expression of DsbA-L in renal tubules of patients with DN significantly decreased and was positively correlated with peroxisomal function. Taken together, these results highlight an important role of DsbA-L in ameliorating tubular injury in DN by improving peroxisomal function.

Celastrol Combats Methicillin-Resistant Staphylococcus aureus by Targeting Δ1 -Pyrroline-5-Carboxylate Dehydrogenase.

The emergence and rapid spread of methicillin-resistant Staphylococcus aureus (MRSA) raise a critical need for alternative therapeutic options. New antibacterial drugs and targets are required to combat MRSA-associated infections. Based on this study, celastrol, a natural product from the roots of Tripterygium wilfordii Hook. f., effectively combats MRSA in vitro and in vivo. Multi-omics analysis suggests that the molecular mechanism of action of celastrol may be related to Δ1 -pyrroline-5-carboxylate dehydrogenase (P5CDH). By comparing the properties of wild-type and rocA-deficient MRSA strains, it is demonstrated that P5CDH, the second enzyme of the proline catabolism pathway, is a tentative new target for antibacterial agents. Using molecular docking, bio-layer interferometry, and enzyme activity assays, it is confirmed that celastrol can affect the function of P5CDH. Furthermore, it is found through site-directed protein mutagenesis that the Lys205 and Glu208 residues are key for celastrol binding to P5CDH. Finally, mechanistic studies show that celastrol induces oxidative stress and inhibits DNA synthesis by binding to P5CDH. The findings of this study indicate that celastrol is a promising lead compound and validate P5CDH as a potential target for the development of novel drugs against MRSA.

Cryo-EM structures of ClC-2 chloride channel reveal the blocking mechanism of its specific inhibitor AK-42.

ClC-2 transports chloride ions across plasma membranes and plays critical roles in cellular homeostasis. Its dysfunction is involved in diseases including leukodystrophy and primary aldosteronism. AK-42 was recently reported as a specific inhibitor of ClC-2. However, experimental structures are still missing to decipher its inhibition mechanism. Here, we present cryo-EM structures of apo ClC-2 and its complex with AK-42, both at 3.5 Å resolution. Residues S162, E205 and Y553 are involved in chloride binding and contribute to the ion selectivity. The side-chain of the gating glutamate E205 occupies the putative central chloride-binding site, indicating that our structure represents a closed state. Structural analysis, molecular dynamics and electrophysiological recordings identify key residues to interact with AK-42. Several AK-42 interacting residues are present in ClC-2 but not in other ClCs, providing a possible explanation for AK-42 specificity. Taken together, our results experimentally reveal the potential inhibition mechanism of ClC-2 inhibitor AK-42.

Gallic Acid Restores the Sulfonamide Sensitivity of Multidrug-Resistant Streptococcus suis via Polypharmaceology Mechanism.

Due to the large amount of antibiotics used for human therapy, agriculture, and even aquaculture, the emergence of multidrug-resistant Streptococcus suis (S. suis) led to serious public health threats. Antibiotic-assisted strategies have emerged as a promising approach to alleviate this crisis. Here, the polyphenolic compound gallic acid was found to enhance sulfonamides against multidrug-resistant S. suis. Mechanistic analysis revealed that gallic acid effectively disrupts the integrity and function of the cytoplasmic membrane by dissipating the proton motive force of bacteria. Moreover, we found that gallic acid regulates the expression of dihydrofolate reductase, which in turn inhibits tetrahydrofolate synthesis. As a result of polypharmacology, gallic acid can fully restore sulfadiazine sodium activity in the animal infection model without any drug resistances. Our findings provide an insightful view into the threats of antibiotic resistance. It could become a promising strategy to resolve this crisis.

Bowman's capsule rupture and the Oxford MEST-C score in adult patients with Immunoglobulin a vasculitis nephritis: a single center experience.

OBJECTIVE: Bowman's capsule rupture (BCR) is a glomerular pathological change, but it is still not well recognized in immunoglobulin A vasculitis nephritis (IgAV-N). The Oxford MEST-C score is a classification for IgA nephropathy; however, its clinical correlation and prognostic value in adult patients with IgAV-N are unclear. METHODS: A retrospective study of 145 adult patients with IgAV-N diagnosed by renal biopsy was conducted. Clinical manifestations, pathological changes and the prognosis of IgAV-N patients were compared depending on the presence or absence of BCR, International Study of Kidney Disease in Children (ISKDC) classification and MEST-C score. The primary endpoint events were end-stage renal disease, renal replacement therapy and all-cause death. RESULTS: In total, 51 of 145 (35.17%) patients with IgAV-N presented with BCR. Patients with BCR had more proteinuria, lower serum albumin, and more crescents. Compared with IgAV-N patients with crescents only, 51/100 patients with crescents combined with BCR had a higher proportion of crescents in all glomeruli (15.79% vs. 9.09%; p = 0.003). Patients with higher ISKDC grades had more severe clinical presentation, but it did not reflect the prognosis. However, the MEST-C score not only reflected clinical manifestations but also predicted prognosis (p < 0.05). BCR contributed to the effectiveness of the MEST-C score in predicting the prognosis of IgAV-N (C-index: 0.845 to 0.855). CONCLUSIONS: BCR is associated with clinical manifestations and pathological changes in patients with IgAV-N. The ISKDC classification and MEST-C score are related to the patient's condition, but only the MEST-C score is correlated with the prognosis of patients with IgAV-N, while BCR can improve its predictive ability.

BCR was associated with clinical manifestations and pathological changes in patients with IgAV-N, particularly crescents.The ISKDC classification was related to clinical manifestations of patients with IgAV-N, but it wasn't associated with prognosis.The Oxford MEST-C score was correlated to clinical presentations and prognosis of patients with IgAV-N, while BCR can improve its predictive ability.

Structure-Based Redesign of a Methanol Oxidase into an "Aryl Alcohol Oxidase" for Enzymatic Synthesis of Aromatic Flavor Compounds.

Alcohol oxidases (AOxs) catalyze the aerobic oxidation of alcohols to the corresponding carbonyl products (aldehydes or ketones), producing only H2O2 as the byproduct. The majority of known AOxs, however, have a strong preference for small, primary alcohols, limiting their broad applicability, e.g., in the food industry. To broaden the product scope of AOxs, we performed structure-guided enzyme engineering of a methanol oxidase from Phanerochaete chrysosporium (PcAOx). The substrate preference was extended from methanol to a broad range of benzylic alcohols by modifying the substrate binding pocket. A mutant (PcAOx-EFMH) with four substitutions exhibited improved catalytic activity toward benzyl alcohols with increased conversion and kcat toward the benzyl alcohol from 11.3 to 88.9% and from 0.5 to 2.6 s-1, respectively. The molecular basis for the change of substrate selectivity was analyzed by molecular simulation.

Modeling of Olfactory Receptors.

Olfactory receptors (ORs) form the largest subfamily within class A G protein-coupled receptors (GPCRs). No experimental structural data of any OR is available to date. Homology modeling has become a popular strategy to propose plausible OR models, in order to study the structure-function relationships of the receptors and to aid the discovery and development of ligands capable of modulating receptor activity. In this chapter, we provide a general guideline for OR structure construction, including the collection of candidate templates, structure-based sequence alignment, 3D structure construction, ligand docking, and molecular dynamic simulation.