Stephanie NguyenBlagojce JovcevskiJia Q. TruongTara L. Pukala...
12页
查看更多>>摘要：Abstract The metabolic enzyme, enolase, plays a crucial role in the cytoplasm where it maintains cellular energy production within the process of glycolysis. The main role of enolase in glycolysis is to convert 2‐phosphoglycerate to phosphoenolpyruvate; however, enolase can fulfill roles that deviate from this function. In pathogenic bacteria and fungi, enolase is also located on the cell surface where it functions as a virulence factor. Surface‐expressed enolase is a receptor for human plasma proteins, including plasminogen, and this interaction facilitates nutrient acquisition and tissue invasion. A novel approach to developing antifungal drugs is to inhibit the formation of this complex. To better understand the structure of enolase and the interactions that may govern complex formation, we have solved the first X‐ray crystal structure of enolase from Aspergillus fumigatus (2.0??) and have shown that it preferentially adopts a dimeric quaternary structure using native mass spectrometry. Two additional X‐ray crystal structures of A. fumigatus enolase bound to the endogenous substrate 2‐phosphoglycerate and product phosphoenolpyruvate were determined and kinetic characterization was carried out to better understand the details of its canonical function. From these data, we have produced a model of the A. fumigatus enolase and human plasminogen complex to provide structural insights into the mechanisms of virulence and aid future development of small molecules or peptidomimetics for antifungal drug design.
查看更多>>摘要：Abstract Protein adaptations to extreme environmental conditions are drivers in biotechnological process optimization and essential to unravel the molecular limits of life. Most proteins with such desirable adaptations are found in extremophilic organisms inhabiting extreme environments. The deep sea is such an environment and a promising resource that poses multiple extremes on its inhabitants. Conditions like high hydrostatic pressure and high or low temperature are prevalent and many deep‐sea organisms tolerate multiple of these extremes. While molecular adaptations to high temperature are comparatively good described, adaptations to other extremes like high pressure are not well‐understood yet. To fully unravel the molecular mechanisms of individual adaptations it is probably necessary to disentangle multifactorial adaptations. In this study, we evaluate differences of protein structures from deep‐sea organisms and their respective related proteins from nondeep‐sea organisms. We created a data collection of 1281 experimental protein structures from 25 deep‐sea organisms and paired them with orthologous proteins. We exhaustively evaluate differences between the protein pairs with machine learning and Shapley values to determine characteristic differences in sequence and structure. The results show a reasonable discrimination of deep‐sea and nondeep‐sea proteins from which we distinguish correlations previously attributed to thermal stability from other signals potentially describing adaptions to high pressure. While some distinct correlations can be observed the overall picture appears intricate.
Wanlei WeiChristopher R. CorbeilFrancis GaudreaultChristophe Deprez...
9页
查看更多>>摘要：Abstract Antibody‐based therapeutics for treatment of various tumors have grown rapidly in recent years. Unfortunately, safety issues, attributed to off‐tumor effects and cytotoxicity, are still a significant concern with the standard of care. Improvements to ensure targeted delivery of antitumor pharmaceuticals are desperately needed. We previously demonstrated that incorporating histidyl pH‐switches in an anti‐HER2 antibody induced selective antigen binding under acidic pH conditions (MAbs 2020;12:1682866). This led to an improved safety profile due to preferential targeting of the oncoprotein in the acidic solid tumor microenvironment. Following this success, we expanded this approach to a set of over 400 antibody structures complexed with over 100 different human oncoproteins, associated with solid tumors. Calculations suggested that mutations to His of certain residue types, namely Trp, Arg, and Tyr, could be significantly more successful for inducing pH‐dependent binding under acidic conditions. Furthermore, 10 positions within the complementarity‐determining region were also predicted to exhibit greater successes. Combined, these two accessible metrics could serve as the basis for a sequence‐based engineering of pH‐selective binding. This approach could be applied to most anticancer antibodies, which lack detailed structural characterization.
查看更多>>摘要：Abstract Glutathione (GSH) is synthesized in two ATP‐dependent reactions by glutamate‐cysteine ligase (Gcl) and glutathione synthetase (Gs). Myxococcus xanthus, a gram‐negative bacterium belonging to δ‐proteobacteria, possesses mxGcl and mxGs, which have high sequence identity with the enzymes from plants and bacteria, respectively. MxGcl2 was activated by Mn2+, but not by Mg2+, and stabilized in the presence of 5?mM Mn2+ or Mg2+. Sequence comparison of mxGcl2 and Brassica juncea Gcl indicated that they have the same active site residues, except for Tyr330, which interacts with Cys and which in mxGcl2 is represented by Leu267. The substitution of Leu267 with Tyr resulted in the loss of mxGcl2 activity, but that with Met (found in cyanobacterial Gcls) increased the mxGcl2 affinity for Cys. GSH and its oxidized form GSSG equally inhibited the activity of mxGcl2; the inhibition was augmented by ATP at concentrations >3?mM. Buthionine sulfoximine inactivated mxGcl2 with Ki?=?2.1?μM, which was lower than those for Gcls from other organisms. The mxGcl2 activity was also suppressed by pyrophosphate and polyphosphates. MxGs was a dimer, and its activity was induced by Mg2+ but strongly inhibited by Mn2+ even in the presence of 10?mM?Mg2+. MxGs was inhibited by GSSG at Ki?=?3.6?mM. Approximately 1?mM GSH was generated with 3?units of mxGcl2 and 6?units of mxGs from 5?mM Glu, Cys, and Gly, and 10?mM ATP. Our results suggest that GSH production in M. xanthus mostly depends on mxGcl2 activity.
查看更多>>摘要：Abstract The binding channel of Schistosoma glutathione transferase (SGST) has been identified to possess a non‐substrate site implicated in enzyme inhibition. This binding channel is formed by the interface of the GST dimer. We produced a comparative characterization of the SGST dimer interface with respect to that of human GST (hGST) analogues using the selective binding of bromosulfophthalein (BSP). First, two SGST and three hGST structures were used as search queries to assemble a data set of 48 empirical GST structures. Sequence alignment to generate a universal residue indexing scheme was then performed, followed by local superposition of the dimer interface. Principal component analysis revealed appreciable variation of the dimer interface, suggesting the potential for selective inhibition of SGST. BSP was found to dock invariably in the dimer interface core pocket, placing it in proximity to the GST catalytic domains, through which it may exert its inhibitory behavior. Binding poses across the GST forms were distinguished with ligand interaction profiling, where SGST complexes showed stabilization of ligand aromatic‐ and sulfonate moieties, which altogether anchor the ligand and produce a tight association. In comparison, missing aromatic stabilization in the hGST complexes impart large bonding distances, causing mobile poses likely to dissociate. Altogether, this study illustrates the potential for selective inhibition of SGST, rationalizes the selective behavior of the BSP inhibitor, and produces a reliable metric for construction and validation of pharmacophore models of the SGST binding channel.
查看更多>>摘要：Abstract Three‐dimensional structures of I86A and C295A mutant secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter pseudoethanolicus were determined by x‐ray crystallography. The tetrameric structure of C295A‐SADH soaked with NADP+ and dimethyl sulfoxide (DMSO) was determined to 1.85?? with an Rfree of 0.225. DMSO is bound to the tetrahedral zinc in each subunit, with ligands from SG of Cys‐37, NE2 of His‐59, and OD2 of Asp‐150. The nicotinamide ring of NADP is hydrogen‐bonded to the N of Ala‐295 and the O of Val‐265 and Gly‐293. The O of DMSO is connected to a network of hydrogen bonds with OG of Ser‐39, the 3′‐OH of NADP, and ND1 of His‐42. The structure of I86A‐SADH soaked with 2‐pentanol and NADP+ contains (R)‐2‐pentanol bound in each subunit, ligated to the tetrahedral zinc, and connected to the proton relay network. The structure of I86A‐SADH soaked with 3‐methylcyclohexanol and NADP+ has alcohol bound in three subunits. Two of the sites have the alcohol ligated to the zinc in an axial position, with OE2 of Glu‐60 in the other axial position of a trigonal bipyramidal complex. One site has 3‐methylcyclohexanol bound noncovalently, with the zinc in an inverted tetrahedral geometry with Glu‐60. The fourth site also has the zinc in a trigonal bipyramidal complex with axial Glu‐60 and water ligands. These structures demonstrate that ligand exchange of SADH involves pentacoordinate and inverted zinc complexes with Glu‐60. Furthermore, we see a network of hydrogen bonds connecting the substrate oxygen to the external solvent that is likely to play a role in the mechanism of SADH.
Penelope‐Marie B. ClanorChristine N. BuchholzJonathan E. HayesMichael A. Friedman...
10页
查看更多>>摘要：Abstract The cone‐rod homeobox (CRX) protein is a critical K50 homeodomain transcription factor responsible for the differentiation and maintenance of photoreceptor neurons in the vertebrate retina. Mutant alleles in the human gene encoding CRX result in a variety of distinct blinding retinopathies, including retinitis pigmentosa, cone‐rod dystrophy, and Leber congenital amaurosis. Despite the success of using in vitro biochemistry, animal models, and genomics approaches to study this clinically relevant transcription factor over the past 25?years since its initial characterization, there are no high‐resolution structures in the published literature for the CRX protein. In this study, we use bioinformatic approaches and small‐angle X‐ray scattering (SAXS) structural analysis to further understand the biochemical complexity of the human CRX homeodomain (CRX‐HD). We find that the CRX‐HD is a compact, globular monomer in solution that can specifically bind functional cis‐regulatory elements encoded upstream of retina‐specific genes. This study presents the first structural analysis of CRX, paving the way for a new approach to studying the biochemistry of this protein and its disease‐causing mutant protein variants.
查看更多>>摘要：Abstract Acinetobacter baumannii is a Gram‐negative bacterium commonly found in soil and water that can cause human infections of the blood, lungs, and urinary tract. Of particular concern is its prevalence in health‐care settings where it can survive on surfaces and shared equipment for extended periods of time. The capsular polysaccharide surrounding the organism is known to be the major contributor to virulence. The structure of the K57 capsular polysaccharide produced by A. baumannii isolate BAL_212 from Vietnam was recently shown to contain the rare sugar 4‐acetamido‐4,6‐dideoxy‐d‐glucose. Three enzymes are required for its biosynthesis, one of which is encoded by the gene H6W49_RS17300 and referred to as VioB, a putative N‐acetyltransferase. Here, we describe a combined structural and functional analysis of VioB. Kinetic analyses show that the enzyme does, indeed, function on dTDP‐4‐amino‐4,6‐dideoxy‐d‐glucose with a catalytic efficiency of 3.9 x 104?M?1?s?1 (±6000), albeit at a reduced value compared to similar enzymes. Three high‐resolution X‐ray structures of various enzyme/ligand complexes were determined to resolutions of 1.65?? or better. One of these models represents an intermediate analogue of the tetrahedral transition state. Differences between the VioB structure and those determined for the N‐acetyltransferases from Campylobacter jejuni (PglD), Caulobacter crescentus (PerB), and Psychrobacter cryohalolentis (Pcryo_0637) are highlighted. Taken together, this investigation sheds new insight into the Type I sugar N‐acetyltransferases.
查看更多>>摘要：Abstract Intrinsically disordered protein (IDP) plays an important role in liquid–liquid phase separation (LLPS). RNA‐binding protein fused in sarcoma (FUS) is a well‐studied IDP that induces LLPS since its low‐complexity core region (FUS‐LC‐core) is essential for droplet formation through contacts between FUS‐LC‐cores. Several experimental studies have reported that adenosine triphosphate (ATP) concentrations modulate LLPS‐driven droplet formation through the dissolution of FUS. To elucidate the role of ATP in this dissolution, microsecond‐order all‐atom molecular dynamics (MD) simulations were performed for a crowded system of FUS‐LC‐cores in the presence of multiple ATP molecules. Our analysis revealed that the adenine group of ATP frequently contacted the FUS‐LC‐core, and the phosphoric acid group of ATP was exposed to the external solvent, which promoted both hydration and solubilization of FUS.
NSTL
Wiley