Pocket Of Residues Research Articles

Molecular Dynamics Simulation Reveal the Structure-Activity Relationships of Kainoid Synthases.

Kainoid synthases are key enzymes in the biosynthesis of kainoids. Kainoids, as represented by DA and KA, are a class of naturally occurring non-protein amino acids with strong neurotransmitter activity in the mammalian central nervous system. Marine algae kainoid synthases include PnDabC from diatoms, which synthesizes domoic acid (DA), and DsKabC and GfKabC from red algae, which synthesize kainic acid (KA). Elucidation of the catalytic mechanism of kainoid synthases is of great significance for the rational design of better biocatalysts to promote the industrial production of kainoids for use in new drugs. Through modeling, molecular docking, and molecular dynamics simulations, we investigated the conformational dynamics of kainoid synthases. We found that the kainoid synthase complexes showed different stability in the simulation, and the binding and catalytic processes showed significant conformational transformations of kainoid synthase. The residues involved in specific interactions with the substrate contributed to the binding energy throughout the simulation process. Binding energy, the relaxed active pocket, electrostatic potential energy of the active pocket, the number and rotation of aromatic residues interacting with substrates during catalysis, and the number and frequency of hydrogen bonds between the individual functional groups revealed the structure-activity relationships and affected the degree of promiscuity of kainoid synthases. Our research enriches the understanding of the conformational dynamics of kainoid synthases and has potential guiding significance for their rational design.

Comparative analysis defines a broader FMRFamide-gated sodium channel family and determinants of neuropeptide sensitivity

FMRFamide (Phe-Met-Arg-Phe-amide, FMRFa) and similar neuropeptides are important physiological modulators in most invertebrates, but the molecular basis of FMRFa activity at its receptors is unknown. We therefore sought to identify the molecular determinants of FMRFa potency against one of its native targets, the excitatory FMRFa-gated sodium channel (FaNaC) from gastropod mollusks. Using molecular phylogenetics and electrophysiological measurement of neuropeptide activity, we identified a broad FaNaC family that includes mollusk and annelid channels gated by FMRFa, FVRIamides, and/or Wamides (or myoinhibitory peptides). A comparative analysis of this broader FaNaC family and other channels from the overarching degenerin (DEG)/epithelial sodium channel (ENaC) superfamily, incorporating mutagenesis and experimental dissection of channel function, identified a pocket of amino acid residues that determines activation of FaNaCs by neuropeptides. Although this pocket has diverged in distantly related DEG/ENaC channels that are activated by other ligands but enhanced by FMRFa, such as mammalian acid-sensing ion channels, we show that it nonetheless contains residues that determine enhancement of those channels by similar peptides. This study thus identifies amino acid residues that determine FMRFa neuropeptide activity at FaNaC receptor channels and illuminates the evolution of ligand recognition in one branch of the DEG/ENaC superfamily of ion channels.

The impact of curcumin derived polyphenols on the structure and flexibility COVID-19 main protease binding pocket: a molecular dynamics simulation study.

The newly occurred SARS-CoV-2 caused a leading pandemic of coronavirus disease (COVID-19). Up to now it has infected more than one hundred sixty million and killed more than three million people according to 14 May 2021 World Health Organization report. So far, different types of studies have been conducted to develop an anti-viral drug for COVID-19 with no success yet. As part of this, silico were studied to discover and introduce COVID-19 antiviral drugs and results showed that protease inhibitors could be very effective in controlling. This study aims to investigate the binding affinity of three curcumin derived polyphenols against COVID-19 the main protease (Mpro), binding pocket, and identification of important residues for interaction. In this study, molecular modeling, auto-dock coupled with molecular dynamics simulations were performed to analyze the conformational, and stability of COVID-19 binding pocket with diferuloylmethane, demethoxycurcumin, and bisdemethoxycurcumin. All three compounds have shown binding affinity −39, −89 and −169.7, respectively. Demethoxycurcumin and bisdemethoxycurcumin showed an optimum binding affinity with target molecule and these could be one of potential ligands for COVID-19 therapy. And also, COVID-19 main protease binding pocket binds with the interface region by one hydrogen bond. Moreover, the MD simulation parameters indicated that demethoxycurcumin and bisdemethoxycurcumin were stable during the simulation run. These findings can be used as a baseline to develop therapeutics with curcumin derived polyphenols against COVID-19.

Macrolactin A as a Novel Inhibitory Agent for SARS-CoV-2 Mpro: Bioinformatics Approach.

COVID-19 is a disease that puts most of the world on lockdown and the search for therapeutic drugs is still ongoing. Therefore, this study used in silico screening to identify natural bioactive compounds from fruits, herbaceous plants, and marine invertebrates that are able to inhibit protease activity in SARS-CoV-2 (PDB: 6LU7). We have used extensive screening strategies such as drug likeliness, antiviral activity value prediction, molecular docking, ADME, molecular dynamics (MD) simulation, and MM/GBSA. A total of 17 compounds were shortlisted using Lipinski’s rule in which 5 compounds showed significant predicted antiviral activity values. Among these 5, only 2 compounds, Macrolactin A and Stachyflin, showed good binding energy of −9.22 and −8.00 kcal/mol, respectively, within the binding pocket of the Mpro catalytic residues (HIS 41 and CYS 145). These two compounds were further analyzed to determine their ADME properties. The ADME evaluation of these 2 compounds suggested that they could be effective in developing therapeutic drugs to be used in clinical trials. MD simulations showed that protein–ligand complexes of Macrolactin A and Stachyflin with the target receptor (6LU7) were stable for 100 nanoseconds. The MM/GBSA calculations of Mpro–Macrolactin A complex indicated higher binding free energy (−42.58 ± 6.35 kcal/mol). Dynamic cross-correlation matrix (DCCM) and principal component analysis (PCA) on the residual movement in the MD trajectories further confirmed the stability of Macrolactin A bound state with 6LU7. In conclusion, this study showed that marine natural compound Macrolactin A could be an effective therapeutic inhibitor against SARS-CoV-2 protease (6LU7). Additional in vitro and in vivo validations are strongly needed to determine the efficacy and therapeutic dose of Macrolactin A in biological systems.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12010-021-03608-7.

Naturally‐Occurring Variants of Muscle‐Type Creatine Kinase Exhibit Altered Tenofovir Monophosphate Phosphorylation Activity

Tenofovir (TFV), prescribed as a disoproxil fumarate prodrug, is a component of the only FDA‐approved regimen for the prevention of HIV. TFV is an acyclic nucleotide analog reverse transcriptase inhibitor prodrug that mimics a nucleotide monophosphate in structure and must be phosphorylated twice by intracellular kinases to produce the pharmacologically active TFV‐diphosphate (DP) form. There have been clinical observations of variation in intracellular TFV‐DP levels, along with efficacy of TFV‐based HIV pre‐exposure prophylaxis (PrEP). We hypothesize that interindividual differences in the kinases that phosphorylate TFV may be a cause of these outcomes. We have elucidated the kinases responsible for TFV phosphorylation in tissues important to forming a barrier of protection against HIV infection, including muscle‐type creatine kinase (CKM), which is the primary kinase responsible for the phosphorylation of TFV‐monophosphate (MP) to the active TFV‐DP in colon tissue. In pursuit of understanding interindividual differences in these kinases, we sequenced the CKM gene in over 1000 patient samples and discovered several previously unreported single nucleotide variants, some of which lead to mutations in the CKM amino acid sequence. Using bioinformatic tools, we predicted that several of these mutants may markedly decrease enzyme function toward the endogenous substrate of the enzyme, ADP. We performed molecular docking simulations, using AutoDock, to model the binding of TFV‐MP to CKM and found that TFV‐MP binds to CKM much as does ADP, which TFV‐MP mimics in structure, suggesting that the bioinformatic predictions may also apply to changes in TFV‐MP phosphorylation activity of CKM. Docking results reveal a pocket of active site arginine residues at positions 130, 132, 236, 292, and 320 as likely important to charge stabilization of the phosphates of TFV‐MP. Notably, our sequencing revealed genetic variants producing R130H and R132C mutations that are predicted to decrease enzyme activity. In order to determine the effects of the naturally‐occurring point mutations on CKM activity toward TFV‐MP, wild‐type human CKM and 15 mutants from the genetic variation analysis were recombinantly expressed and purified. Then, in vitro assays were performed by incubating the proteins with TFV‐MP and measuring TFV‐DP formation via ultra‐high performance liquid chromatography tandem mass spectrometry. These results reveal 11 mutations in CKM that significantly decrease TFV‐MP phosphorylation. Three active site mutations, R130H, R132C, and N286I, along with W211R resulted in a decrease in TFV‐DP formation to only 3% of that of wild‐type CKM. Additionally, T35I, R43Q, I92M, H97Y, F169L, and Y173C mutations decreased activity to 14–25% of that of wild‐type. This evidence suggests that CKM genetics may play a role in interindividual variability in TFV activation and potentially the efficacy of TFV in PrEP settings.Support or Funding InformationThis research is funded by NIH R01 AI128781

Effect of mutations in porcine CYB5A and CYP17A1 on the metabolism of pregnenolone

Cytochrome P450 17A1 (CYP17A1) catalyses the 17α-hydroxylation and 17,20 lyase reactions to convert pregnenolone to 17α-hydroxypregnenolone (17OHP) and subsequently the androgen dehydroepiandrosterone (DHEA). In pigs and humans, CYP17A1 also catalyses the delta-16-synthase reaction to produce the 16-androstene steroid 5,16-androstadien-3β-ol (16A), which is then further metabolised to the sex pheromone androstenone. Cytochrome b5A (CYB5A) stimulates the 17,20 lyase reaction and is required for the delta 16-synthase reaction. We have identified and mutated residues in porcine CYP17A1 and CYB5A that may alter the synthesis of DHEA and 16A. This included residues in the steroid binding pocket of CYP17A1 and residues on the surface of CYP17A1 and CYB5A that are involved in binding of CYP17A1 to CYB5A. We then expressed the various mutations of CYB5A and CYP17A1 along with porcine cytochrome P450 oxidoreductase (POR) and cytochrome b5 reductase (CYB5R3) in HEK293 cells and measured the formation of metabolites 16A, 17OHP and DHEA from radiolabelled pregnenolone by high performance liquid chromatography (HPLC). Mutations were identified in both CYP17A1 and CYB5A that affected the production of the different metabolites and also affected the overall production of metabolites. Several combinations of mutations decreased the production of both 16A and DHEA and increased production of 17OHP, while the N62S mutation of CYB5A with wild type CYP17A1 increased production of both 16A and DHEA. The best combination of mutations to reduce the production of 16A, while maintaining the production of DHEA and the overall conversion similar to wild type are the N21K, L28V, N21K/L28V and the R52 M/N62S mutations of CYB5A with the D103S mutation of CYP17A1.

In silico insights on tankyrase protein: A potential target for colorectal cancer

The Wnt/β-catenin pathway plays an important regulatory role in cancer signaling and cell regenerative mechanisms. Its suppression has long been considered as an important challenge of anticancer treatment and management. The poly(ADP-ribose) polymerase (PARP) family represented as a new class of therapeutic targets with diverse potential disease indications. Tankyrase (TNKS) is considered to be a potential target for the intervention of various cancers. The main objective of the work is to explore the molecular and quantum mechanics of the drug-like compounds and to identify the potential inhibitors for TNKS protein using the structure and ligand-based virtual screening from several databases and to explore the binding pocket and interactions of active residues. The screened compounds were further filtered using binding-free energy calculation and molecular dynamics simulation studies. The results have provided a strong molecular knowledge of TNKS and offered top hit potent inhibitors. The identified lead compounds LC_40781, LC_40777, LC_39767, LC_8346, NCI_682438, and NCI_721141 were observed to have potent activity against TNKS protein. The hydrogen bonding of compounds with Asp1198, His1201, Tyr1203 in TNKS1 and Gly1032, Ser1068 in TNKS2 are the key interactions plays a major role in binding energy. Therefore, the outcome of the study would help for further validation and provides valuable information to guide the future TNKS-specific inhibitor designing.Communicated by Ramaswamy H. Sarma

Modeling, molecular docking, probing catalytic binding mode of acetyl-CoA malate synthase G in Brucella melitensis 16M

There are enormous evidences and previous reports standpoint that the enzyme of glyoxylate pathway malate synthase G (MSG) is a potential virulence factor in several pathogenic organisms, including Brucella melitensis 16M. Where the lack of crystal structures for best candidate proteins like MSG of B. melitensis 16M creates big lacuna to understand the molecular pathogenesis of brucellosis. In the present study, we have constructed a 3-D structure of MSG of Brucella melitensis 16M in MODELLER with the help of crystal structure of Mycobacterium tuberculosis malate synthase (PDB ID: 2GQ3) as template. The stereo chemical quality of the restrained model was evaluated by SAVES server; remarkably we identified the catalytic functional core domain located at 4th cleft with conserved catalytic amino acids, start at ILE 59 to VAL 586 manifest the function of the protein. Furthermore, virtual screening and docking results reveals that best leadmolecules binds at the core domain pocket of MSG catalytic residues and these ligand leads could be the best prospective inhibitors to treat brucellosis.

Tubulation by Amphiphysin Requires Concentration-Dependent Switching from Wedging to Scaffolding

BAR proteins are involved in a variety of membrane remodeling events but how they can mold membranes into different shapes remains poorly understood. Using electron paramagnetic resonance, wefind that vesicle binding of the N-BAR protein amphiphysin is predominantly mediated by the shallow insertion of amphipathic N-terminal helices. In contrast, the interaction with tubes involves deeplyinserted N-terminal helices together with the concave surface of the BAR domain, which acts as a scaffold. Combined with the observed concentration dependence of tubulation and BAR domain scaffolding, the data indicate that initial membrane deformations and vesicle binding are mediated by insertion of amphipathic helical wedges, while tubulation requires high protein densities at which oligomeric BAR domain scaffolds form. In addition, we identify a pocket of residues on the concave surface of the BAR domain that insert deeply into tube membrane. Interestingly, this pocket harbors a number of disease mutants in the homologous amphiphysin 2.

The solution structure of human calcium-bound S100A4 mutated at four cysteine loci.

The S100 proteins constitute the largest family within the EF-hand protein superfamily, shown to bind and control various proteins involved in several cellular functions such as proliferation, differentiation, apoptosis, Ca homeostasis, and energy metabolism (Donato et al. 2013; Hermann et al. 2012; Leclerc and Heizmann 2011). They regulate a wide range of important cellular processes via protein–protein interactions (Schafer and Heizmann 1996). Calcium interactions with the EF-hand motifs result in a conformation change of target protein binding by exposing hydrophobic regions in S100 proteins (Smith and Shaw 1998). The calcium binding EF-hand motif starts the action with structural changes in the S100 proteins, allowing them to interact through target selectivity (Yap et al. 1999; Zimmer and Weber 2010). The protein S100A4 was first derived from both tumor and stroma. It is a homodimeric protein in solution and has been shown to function as a metastasis-promoting protein (Ambartsumian et al. 2005; Ismail et al. 2010). Its presence has now been well documented in many cancers including breast, colorectal, gastric, pancreatic, and bladder cancers. It plays a role in tumor formation and angiogenesis (Ambartsumian et al. 2001; Barraclough et al. 2009; Ford and Zain 1995). The hinge region and C-terminal EF-hand of the S100A4 protein are unique compared to other S100 proteins; however most S100 proteins are involved in target protein binding. The binding of calcium ions produces conformational changes in proteins resulting in exposure of the hydrophobic pocket of residues in helices 3 and 5, the hinge region, and the C-terminal EF-hand (Malashkevich et al. 2008; Mishra et al. 2012; Semov et al. 2005). Previous results have provided insights into the dynamic mechanism of the C-terminal in S100A4 as a mediator of S100A4-driven metastasis, and they highlight its role in tuning the Ca-binding affinity of S100A4. These results also suggest that locking the C-terminus to the core domain may be an alternative strategy for inhibiting its metastasispromoting activities (Duelli et al. 2014). In this study, we mutated four cysteine residues (Cys3, Cys76, Cys81, and Cys86) on S100A4 to serine. The Cys3 residue is located very near the N-terminus, and the remaining three are in helix 4 (H4) (Pathuri et al. 2008). We labeled this protein ‘mutant S100A4’ and determined the three-dimensional structure of the calcium-bound of this protein using NMR. With this structure solved, we could explain why full length S100A4 has weaker calcium-binding affinity than its truncated form, where the last 13 amino acids in the C-terminal are deleted.

Allosteric Switching of Agonist/Antagonist Activity by a Single Point Mutation in the Interluekin-1 Receptor Antagonist, IL-1Ra

The pleiotropic pro-inflammatory cytokine interleukin (IL)-1β has co-evolved with a competitive inhibitor, IL-1 receptor antagonist (IL-1Ra). IL-1β initiates cell signaling by binding the IL-1 receptor (IL-1R) whereas IL-1Ra acts as an antagonist, blocking receptor signaling. The current paradigm for agonist/antagonist functions for these two proteins is based on the receptor–ligand interaction observed in the crystal structures of the receptor–ligand complexes. While IL-1Ra and IL-1β are structurally homologous, IL-1Ra engages only two of the three extracellular domains of the receptor, whereas IL-1β engages all three. We find that an allosteric functional switch exists within a highly conserved pocket of residues, residues 111–120. This region is maintained across all IL-1 family members and serves as a hydrophobic mini-core for IL-1β folding. A key difference across species is a conserved aromatic residue at position 117 in IL-1β, versus a conserved cysteine in IL-1Ra at the analogous position, 116. We find that the replacement of C116 with a phenylalanine switches the protein from an antagonist to an agonist despite the distant location of C116 relative to receptor interaction sites. These results suggest new ways to develop designer cytokine activity into the β-trefoil fold and may be of general use in regulation of this large family of signaling proteins.

Improved Manganese-Oxidizing Activity of DypB, a Peroxidase from a Lignolytic Bacterium

DypB, a dye-decolorizing peroxidase from the lignolytic soil bacterium Rhodococcus jostii RHA1, catalyzes the peroxide-dependent oxidation of divalent manganese (Mn(2+)), albeit less efficiently than fungal manganese peroxidases. Substitution of Asn246, a distal heme residue, with alanine increased the enzyme's apparent k(cat) and k(cat)/K(m) values for Mn(2+) by 80- and 15-fold, respectively. A 2.2 Å resolution X-ray crystal structure of the N246A variant revealed the Mn(2+) to be bound within a pocket of acidic residues at the heme edge, reminiscent of the binding site in fungal manganese peroxidase and very different from that of another bacterial Mn(2+)-oxidizing peroxidase. The first coordination sphere was entirely composed of solvent, consistent with the variant's high K(m) for Mn(2+) (17 ± 2 mM). N246A catalyzed the manganese-dependent transformation of hard wood kraft lignin and its solvent-extracted fractions. Two of the major degradation products were identified as 2,6-dimethoxybenzoquinone and 4-hydroxy-3,5-dimethoxybenzaldehyde, respectively. These results highlight the potential of bacterial enzymes as biocatalysts to transform lignin.

Crystal Structure of the Ectodomain Complex of the CGRP Receptor, a Class-B GPCR, Reveals the Site of Drug Antagonism

Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. CGRP binds to and signals through the CGRP receptor (CGRP-R), a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and potentially modulate the activities of the CLR GPCR subfamily. We also report the structures of CLR/RAMP1 in complex with the clinical receptor antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the peptide-binding cleft at the interface of CLR and RAMP1. These structures illustrate, for the first time, how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases.

Structural basis for dsRNA recognition and interferon antagonism by Ebola VP35

Ebola viral protein 35 (VP35), encoded by the highly pathogenic Ebola virus, facilitates host immune evasion by antagonizing antiviral signaling pathways, including those initiated by RIG-I-like receptors. Here we report the crystal structure of the Ebola VP35 interferon inhibitory domain (IID) bound to short double-stranded RNA (dsRNA), which together with in vivo results reveals how VP35-dsRNA interactions contribute to immune evasion. Conserved basic residues in VP35 IID recognize the dsRNA backbone, whereas the dsRNA blunt ends are 'end-capped' by a pocket of hydrophobic residues that mimic RIG-I-like receptor recognition of blunt-end dsRNA. Residues critical for RNA binding are also important for interferon inhibition in vivo but not for viral polymerase cofactor function of VP35. These results suggest that simultaneous recognition of dsRNA backbone and blunt ends provides a mechanism by which Ebola VP35 antagonizes host dsRNA sensors and immune responses.

Thermodynamic and Hydrodynamic Characterization of the Interaction Between DmsD and the DmsA Twin-Arginine Leader Peptide

The system specific chaperone DmsD plays a role in the maturation of the DMSO reductase enzyme through its interaction with the twin-arginine leader peptide of the catalytic subunit DmsA, prior to its assembly into the holo-enzyme. A pocket of residues, clustered together within the structure of DmsD, has previously been shown to be important for binding a fusion protein composed of 43 of the 45 amino acid residues of the DmsA leader peptide however the region of the DmsA leader peptide that interacts with DmsD has not been identified. Various portions of the DmsA leader peptide were synthesized and assayed for binding to DmsD using isothermal titration calorimetry. A peptide composed of 27 amino acid residues near the C-terminus of the DmsA leader sequence was found to bind to DmsD and subsequently used to characterize the thermodynamics of binding of each of the DmsD variant proteins previously shown to be important for binding to the DmsA leader peptide. Size exclusion chromatography and native-PAGE were used to determine the effect of peptide binding on multimeric state and electrophoretic mobility for each of the variant proteins. In the presence of the peptide, wild type DmsD migrates faster on native-PAGE but remains monomeric while some DmsD variant proteins undergo oligomerization while still changing to a the same faster migrating form on native-PAGE. The electrophoretic mobility, multimeric forms and thermodynamics of peptide binding of mutations in the chaperone leader-binding site are compared.

Aqueous Accessibility to the Transmembrane Regions of Subunit c of the Escherichia coli F1F0 ATP Synthase

Rotary catalysis in F(1)F(0) ATP synthase is powered by proton translocation through the membrane-embedded F(0) sector. Proton binding and release occur in the middle of the membrane at Asp-61 on transmembrane helix (TMH) 2 of subunit c. Previously the reactivity of Cys substituted into TMH2 revealed extensive aqueous access at the cytoplasmic side as probed with Ag(+) and other thiolate-directed reagents. The analysis of aqueous accessibility of membrane-embedded regions in subunit c was extended here to TMH1 and the periplasmic side of TMH2. The Ag(+) sensitivity of Cys substitutions was more limited on the periplasmic versus cytoplasmic side of TMH2. In TMH1, Ag(+) sensitivity was restricted to a pocket of four residues lying directly behind Asp-61. Aqueous accessibility was also probed using Cd(2+), a membrane-impermeant soft metal ion with properties similar to Ag(+). Cd(2+) inhibition was restricted to the I28C substitution in TMH1 and residues surrounding Asp-61 in TMH2. The overall pattern of inhibition, by all of the reagents tested, indicates highest accessibility on the cytoplasmic side of TMH2 and in a pocket of residues around Asp-61, including proximal residues in TMH1. Additionally subunit a was shown to mediate access to this region by the membrane-impermeant probe 2-(trimethylammonium)ethyl methanethiosulfonate. Based upon these results and other information, a pocket of aqueous accessible residues, bordered by the peripheral surface of TMH4 of subunit a, is proposed to extend from the cytoplasmic side of cTMH2 to Asp-61 in the center of the membrane.

Protein surface analysis for function annotation in high‐throughput structural genomics pipeline

Structural genomics (SG) initiatives are expanding the universe of protein fold space by rapidly determining structures of proteins that were intentionally selected on the basis of low sequence similarity to proteins of known structure. Often these proteins have no associated biochemical or cellular functions. The SG success has resulted in an accelerated deposition of novel structures. In some cases the structural bioinformatics analysis applied to these novel structures has provided specific functional assignment. However, this approach has also uncovered limitations in the functional analysis of uncharacterized proteins using traditional sequence and backbone structure methodologies. A novel method, named pvSOAR (pocket and void Surface of Amino Acid Residues), of comparing the protein surfaces of geometrically defined pockets and voids was developed. pvSOAR was able to detect previously unrecognized and novel functional relationships between surface features of proteins. In this study, pvSOAR is applied to several structural genomics proteins. We examined the surfaces of YecM, BioH, and RpiB from Escherichia coli as well as the CBS domains from inosine-5'-monosphate dehydrogenase from Streptococcus pyogenes, conserved hypothetical protein Ta549 from Thermoplasm acidophilum, and CBS domain protein mt1622 from Methanobacterium thermoautotrophicum with the goal to infer information about their biochemical function.

Structural Determinants of Benzodiazepine Allosteric Regulation of GABAAReceptor Currents

Benzodiazepine enhancement of GABA(A) receptor current requires a gamma subunit, and replacement of the gamma subunit by the delta subunit abolishes benzodiazepine enhancement. Although it has been demonstrated that benzodiazepines bind to GABA(A) receptors at the junction between alpha and gamma subunits, the structural basis for the coupling of benzodiazepine binding to allosteric enhancement of the GABA(A) receptor current is unclear. To determine the structural basis for this coupling, the present study used a chimera strategy, using gamma2L-delta GABA(A) receptor subunit chimeras coexpressed with alpha1 and beta3 subunits in human embryonic kidney 293T cells. Different domains of the gamma2L subunit were replaced by delta subunit sequence, and diazepam sensitivity was determined. Chimeric subunits revealed two areas of interest: domain 1 in transmembrane domain 1 (M1) and domain 2 in the C-terminal portion of transmembrane domain 2 (M2) and the M2-M3 extracellular loop. In those domains, site-directed mutagenesis demonstrated that the following two groups of residues were involved in benzodiazepine transduction of current enhancement: residues Y235, F236, T237 in M1; and S280, T281, I282 in M2 as well as the entire M2-M3 loop. These results suggest that a pocket of residues may transduce benzodiazepine binding to increased gating. Benzodiazepine transduction involves a group of residues that connects the N terminus and M1, and another group of residues that may facilitate an interaction between the N terminus and the M2 and M2-M3 loop domains.

PvSOAR: detecting similar surface patterns of pocket and void surfaces of amino acid residues on proteins.

Detecting similar protein surfaces provides an important route for discovering unrecognized or novel functional relationship between proteins. The web server pvSOAR (pocket and void Surfaces Of Amino acid Residues) provides an online resource to identify similar protein surface regions. pvSOAR can take a structure either uploaded by a user or obtained from the Protein Data Bank, and identifies similar surface patterns based on geometrically defined pockets and voids. It provides several search modes to compare protein surfaces by similarity in local sequence, local shape and local orientation. Statistically significant search results are reported for visualization and interactive exploration. pvSOAR can be used to predict biological functions of proteins with known three-dimensional structures but unknown biological roles. It can also be used to study functional relationship between proteins and for exploration of the evolutionary origins of structural elements important for protein function. We present an example using pvSOAR to explore the biological roles of a protein whose structure was solved by the structural genomics project. The pvSOAR web server is available at http://pvsoar.bioengr.uic.edu/.

The nicotinic receptor ligand binding domain.

The ligand binding domain (LBD) of the nicotinic acetylcholine receptor has served as a prototype for understanding molecular recognition in the family of neurotransmitter-gated ion channels. During the past fifty years, studies progressed from fundamental electrophysiological analyses of ACh-evoked ion flow, to biochemical purification of the receptor protein, pharmacological measurements of ligand binding, molecular cloning of receptor subunits, site-directed mutagenesis combined with functional analysis and recently, atomic structural determination. The emerging picture of the nicotinic receptor LBD is a specialized pocket of aromatic and hydrophobic residues formed at interfaces between protein subunits that changes conformation to convert agonist binding into gating of an intrinsic ion channel.