Molecular Dynamics Simulations and Principal Component Analysis on Human Laforin Mutation W32G and W32G/K87A

Mutations in human laforin lead to an autosomal neurodegenerative disorder Lafora disease. In N-terminal carbohydrate binding domain of laforin, two mutations W32G and K87A are reported as highly disease causing laforin mutants. Experimental studies reported that mutations are responsible for the abolishment of glycogen binding which is a critical function of laforin. Our current computational study focused on the role of conformational changes in human laforin structure due to existing single mutation W32G and prepared double mutation W32G/K87A related to loss of glycogen binding. We performed 10 ns molecular dynamics (MD) simulation studies in the Gromacs package for both mutations and analyzed the trajectories. From the results, the global properties like root mean square deviation, root mean square fluctuation, radius of gyration, solvent accessible surface area and hydrogen bonds showed structural changes in atomic level observed in W32G and W32G/K87A laforin mutants. The conformational change induced by mutants influenced the loss of the overall stability of the native laforin. Moreover, the change in overall motion of protein was analyzed by principal component analysis and results showed protein clusters expanded more than native and also change in direction in case of double mutant in conformational space. Overall, our report provides theoretical information on loss of structure–function relationship due to flexible nature of laforin mutants. In conclusion, comparative MD simulation studies support the experimental data on W32G and W32G/K87A related to the lafora disease mechanism on glycogen binding.     

Role of Lys-32 residues in R67 dihydrofolate reductase probed by asymmetric mutations

R67 dihydrofolate reductase (R67 DHFR) is a novel protein encoded by an R-plasmid that confers resistance to the antibiotic, trimethoprim. This homotetrameric enzyme possesses 222 symmetry, which imposes numerous constraints on the single active site pore, including a "one-site-fits-both" strategy for binding its ligands, dihydrofolate (DHF) and NADPH. Previous studies uncovered salt effects on binding and catalysis (Hicks, S. N., Smiley, R. D., Hamilton, J. B., and Howell, E. E. (2003) Biochemistry 42, 10569-10578), however the one or more residues that participate in ionic contacts with the negatively charged tail of DHF as well as the phosphate groups in NADPH were not identified. Several studies predict that Lys-32 residues were involved, however mutations at this residue destabilize the R67 DHFR homotetramer. To study the role of Lys-32 in binding and catalysis, asymmetric K32M mutations have been utilized. To create asymmetry, individual mutations were added to a tandem array of four in-frame gene copies. These studies show one K32M mutation is tolerated quite well, whereas addition of two mutations has variable effects. Two double mutants, K32M:1+2 and K32M: 1+4, which place the mutations on opposite sides of the pore, reduce kcat. However a third double mutant, K32M: 1+3, that places two mutations on the same half pore, enhances kcat 4- to 5-fold compared with the parent enzyme, albeit at the expense of weaker binding of ligands. Because the kcat/Km values for this double mutant series are similar, these mutations appear to have uncovered some degree of non-productive binding. This non-productive binding mode likely arises from formation of an ionic interaction that must be broken to allow access to the transition state. The K32M:1+3 mutant data suggest this interaction is an ionic interaction between Lys-32 and the charged tail of dihydrofolate. This unusual catalytic scenario arises from the 222 symmetry imposed on the single active site pore.     

Molecular dynamics and molecular docking studies on E166A point mutant,R274N/R276N double mutant,and E166A/R274N/R276N triple mutant forms of class A β-lactamases

Bacterial resistance to β-lactams antibiotics is a serious threat to human health. The most common cause of resistance to the β-lactams is the production of β-lactamase that inactivates β-lactams. Specifically, class A extended-spectrum β-lactamase produced by antibiotic resistant bacteria is capable of hydrolyzing extended-spectrum Cephalosporins and Monobactams. Mutations in class A β-lactamases play a crucial role in substrate and inhibitor specificity. In this present study, the E166A point mutant, R274N/R276N double mutant, and E166A/R274N/R276N triple mutant class A β-lactamases are analyzed. Molecular dynamics (MD) simulations are done to understand the consequences of mutations in class A β-lactamases. Root mean square deviation, root mean square fluctuation, radius of gyration, solvent accessibility surface area, hydrogen bond, and essential dynamics analysis results indicate notable loss in stability for mutant class A β-lactamases. MD simulations of native and mutant structures clearly confirm that the substitution of alanine at the position of 166, Asparagine at 274 and 276 causes more flexibility in 3D space. Molecular docking results indicate the mutation in class A β-lactamases which decrease the binding affinity of Cefpirome and Ceftobiprole which are third and fifth generation Cephalosporins, respectively. MD simulation of Ceftobiprole-native and mutant type Class A β-lactamases complexes reveal that E166A/R274N/R276N mutations alter the structure and notable loss in the stability for Ceftobirole-mutant type Class A β-lactamases complexes. Ceftobiprole is currently prescribed for patients with serious bacterial infections; this phenomenon is the probable cause for the effectiveness of Ceftobiprole in controlling bacterial infections.     

Effect of cell polarization on hepatitis C virus entry

The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.     

Mutants of protein kinase A that mimic the ATP-binding site of protein kinase B (AKT)

The mutation of well behaved enzymes in order to simulate less manageable cognates is the obvious approach to study specific features of the recalcitrant target. Accordingly, the prototypical protein kinase PKA serves as a model for many kinases, including the closely related PKB, an AGC family protein kinase now implicated as oncogenic in several cancers. Two residues that differ between the alpha isoforms of PKA and PKB at the adenine-binding site generate differing shapes of the binding surface and are likely to play a role in ligand selectivity. As the corresponding mutations in PKA, V123A would enlarge the adenine pocket, while L173M would alter both the shape and its electronic character of the adenine-binding surface. We have determined the structures of the corresponding double mutant (PKAB2: PKAalpha V123A, L173M) in apo and MgATP-bound states, and observed structural alterations of a residue not previously involved in ATP-binding interactions: the side-chain of Q181, which in native PKA points away from the ATP-binding site, adopts in apo double mutant protein a new rotamer conformation, which places the polar groups at the hinge region in the ATP pocket. MgATP binding forces Q181 back to the position seen in native PKA. The crystal structure shows that ATP binding geometry is identical with that in native PKA but in this case was determined under conditions with only a single Mg ion ligand. Surface plasmon resonance spectroscopy studies show that significant energy is required for this ligand-induced transition. An additional PKA/PKB mutation, Q181K, corrects the defect, as shown both by the crystal structure of triple mutant PKAB3 (PKAalpha V123A, L173M, Q181K) and by surface plasmon resonance spectroscopy binding studies with ATP and three isoquinoline inhibitors. Thus, the triple mutant serves well as an easily crystallizable model for PKB inhibitor interactions. Further, the phenomenon of Q181 shows how crystallographic analysis should accompany mutant studies to monitor possible spurious structural effects.     

Viral kinetics suggests a reconciliation of the disparate observations of the modulation of claudin-1 expression on cells exposed to hepatitis C virus

The tight junction protein claudin-1 (CLDN1) is necessary for hepatitis C virus (HCV) entry into target cells. Recent studies have made disparate observations of the modulation of the expression of CLDN1 on cells following infection by HCV. In one study, the mean CLDN1 expression on cells exposed to HCV declined, whereas in another study HCV infected cells showed increased CLDN1 expression compared to uninfected cells. Consequently, the role of HCV in modulating CLDN1 expression, and hence the frequency of cellular superinfection, remains unclear. Here, we present a possible reconciliation of these disparate observations. We hypothesized that viral kinetics and not necessarily HCV-induced receptor modulation underlies these disparate observations. To test this hypothesis, we constructed a mathematical model of viral kinetics in vitro that mimicked the above experiments. Model predictions provided good fits to the observed evolution of the distribution of CLDN1 expression on cells following exposure to HCV. Cells with higher CLDN1 expression were preferentially infected and outgrown by cells with lower CLDN1 expression, resulting in a decline of the mean CLDN1 expression with time. At the same time, because the susceptibility of cells to infection increased with CLDN1 expression, infected cells tended to have higher CLDN1 expression on average than uninfected cells. Our study thus presents an explanation of the disparate observations of CLDN1 expression following HCV infection and points to the importance of considering viral kinetics in future studies of receptor expression on cells exposed to HCV.     

The localization of FGFR3 mutations causing thanatophoric dysplasia type I differentially affects phosphorylation, processing and ubiquitylation of the receptor

Recurrent missense fibroblast growth factor receptor 3 (FGFR3) mutations have been ascribed to skeletal dysplasias of variable severity including the lethal neonatal thanatophoric dysplasia types I (TDI) and II (TDII). To elucidate the role of activating mutations causing TDI on receptor trafficking and endocytosis, a series of four mutants located in different domains of the receptor were generated and transiently expressed. The putatively elongated X807R receptor was identified as three isoforms. The fully glycosylated mature isoform was constitutively but mildly phosphorylated. Similarly, mutations affecting the extracellular domain (R248C and Y373C) induced moderate constitutive receptor phosphorylation. By contrast, the K650M mutation affecting the tyrosine kinase 2 (TK2) domain produced heavy phosphorylation of the nonglycosylated and mannose-rich isoforms that impaired receptor trafficking through the Golgi network. This resulted in defective expression of the mature isoform at the cell surface. Normal processing was rescued by tyrosine kinase inhibitor treatment. Internalization of the R248C and Y373C mutant receptors, which form stable disulfide-bonded dimers at the cell surface was less efficient than the wild-type, whereas ubiquitylation was markedly increased but apparently independent of the E3 ubiquitin-ligase casitas B-lineage lymphoma (c-Cbl). Constitutive phosphorylation of c-Cbl by the K650M mutant appeared to be related to the intracellular retention of the receptor. Therefore, although mutation K650M affecting the TK2 domain induces defective targeting of the overphosphorylated receptor, a different mechanism characterized by receptor retention at the plasma membrane, excessive ubiquitylation and reduced degradation results from mutations that affect the extracellular domain and the stop codon.     

CD81 and claudin 1 coreceptor association: role in hepatitis C virus entry

Hepatitis C virus (HCV) is an enveloped positive-stranded RNA hepatotropic virus. HCV pseudoparticles infect liver-derived cells, supporting a model in which liver-specific molecules define HCV internalization. Three host cell molecules have been reported to be important entry factors or receptors for HCV internalization: scavenger receptor BI, the tetraspanin CD81, and the tight junction protein claudin-1 (CLDN1). None of the receptors are uniquely expressed within the liver, leading us to hypothesize that their organization within hepatocytes may explain receptor activity. Since CD81 and CLDN1 act as coreceptors during late stages in the entry process, we investigated their association in a variety of cell lines and human liver tissue. Imaging techniques that take advantage of fluorescence resonance energy transfer (FRET) to study protein-protein interactions have been developed. Aequorea coerulescens green fluorescent protein- and Discosoma sp. red-monomer fluorescent protein-tagged forms of CD81 and CLDN1 colocalized, and FRET occurred between the tagged coreceptors at comparable frequencies in permissive and nonpermissive cells, consistent with the formation of coreceptor complexes. FRET occurred between antibodies specific for CD81 and CLDN1 bound to human liver tissue, suggesting the presence of coreceptor complexes in liver tissue. HCV infection and treatment of Huh-7.5 cells with recombinant HCV E1-E2 glycoproteins and anti-CD81 monoclonal antibody modulated homotypic (CD81-CD81) and heterotypic (CD81-CLDN1) coreceptor protein association(s) at specific cellular locations, suggesting distinct roles in the viral entry process.     

R67, the other dihydrofolate reductase: rational design of an alternate active site configuration

R67 dihydrofolate reductase (DHFR) bears no sequence or structural homologies with chromosomal DHFRs. The gene for this enzyme produces subunits that are 78 amino acids long, which assemble into a homotetramer possessing 222 symmetry. More recently, a tandem array of four gene copies linked in-frame was constructed, which produces a monomer containing 312 amino acids named Quad3. Asymmetric mutations in Quad3 have also been constructed to probe the role of Q67 and K32 residues in catalysis. This present study mixes and matches mutations to determine if the Q67H mutation, which tightens binding approximately 100-fold to both dihydrofolate (DHF) and NADPH, can help rescue the K32M mutation. While the latter mutation weakens DHF binding over 60-fold, it concurrently increases kcat by a factor of 5. Two Q67H mutations were added to gene copies 1 and 4 in conjunction with the K32M mutation in gene copies 1 and 3. Addition of these Q67H mutations tightens binding 40-fold, and the catalytic efficiency (kcat/Km(DHF)) of the resulting protein is similar to that of Quad3. Since these Q67H mutations can mostly compensate for the K32M lesion, K32 must not be necessary for DHF binding. Another multimutant combines the K32M mutation in gene copies 1 and 3 with the Q67H mutation in all gene copies. This mutant is inhibited by DHF but not NADPH, indicating that NADPH binds only to the wild type half of the pore, while DHF can bind to either the wild type or mutant half of the pore. This inhibition pattern contrasts with the mutant containing only the Q67H substitution in all four gene copies, which is severely inhibited by both NADPH and substrate. Since gene duplication and divergence are evolutionary tools for gaining function, these constructs are a first step toward building preferences for NADPH and DHF in each half of the active site pore of this primitive enzyme.     

Experimental Evidence for Enhanced Receptor Binding by Rapidly Spreading SARS-CoV-2 Variants

Rapidly spreading new variants of SARS-CoV-2 carry multiple mutations in the viral spike protein which attaches to the angiotensin converting enzyme 2 (ACE2) receptor on host cells. Among these mutations are amino acid changes N501Y (lineage B.1.1.7, first identified in the UK), and the combination N501Y, E484K, K417N (B.1.351, first identified in South Africa), all located at the interface on the receptor binding domain (RBD). We experimentally establish that RBD containing the N501Y mutation results in 7-fold stronger binding to the hACE2 receptor than wild type RBD. The E484K mutation only slightly enhances the affinity for the receptor, while K417N attenuates affinity. As a result, RBD from B.1.351 containing all three mutations binds 3-fold stronger to hACE2 than wild type RBD but 2-fold weaker than N501Y. However, the recently emerging double mutant E484K/N501Y binds even stronger than N501Y. The independent evolution of lineages containing mutations with different effects on receptor binding affinity, viral transmission and immune evasion underscores the importance of global viral genome surveillance and functional characterization.     

Aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in ABCA3 mutants associated with nonfatal pediatric interstitial lung disease

The ATP-binding cassette transporter ABCA3 mediates uptake of choline-phospholipids into intracellular vesicles and is essential for surfactant metabolism in lung alveolar type II cells. We have shown previously that ABCA3 mutations in fatal surfactant deficiency impair intracellular localization or ATP hydrolysis of ABCA3 protein. However, the mechanisms underlying the less severe phenotype of patients with ABCA3 mutation are unclear. In this study, we characterized ABCA3 mutant proteins identified in pediatric interstitial lung disease (pILD). E292V (intracellular loop 1), E690K (adjacent to Walker B motif in nucleotide binding domain 1), and T1114M (8th putative transmembrane segment) mutant proteins are localized mainly in intracellular vesicle membranes as wild-type protein. Lipid analysis and sucrose gradient fractionation revealed that the transport function of E292V mutant protein is moderately preserved, whereas those of E690K and T1114M mutant proteins are severely impaired. Vanadate-induced nucleotide trapping and photoaffinity labeling of wild-type and mutant proteins using 8-azido-[(32)P]ATP revealed an aberrant catalytic cycle in these mutant proteins. These results demonstrate the importance of a functional catalytic cycle in lipid transport of ABCA3 and suggest a pathophysiological mechanism of pILD due to ABCA3 mutation.     

Functional significance of the thyrotropin receptor germline polymorphism D727E

In a toxic thyroid adenoma we identified a novel somatic mutation that constitutively activates the thyrotropin receptor (TSHR). Two heterozygous point mutations at adjacent nucleotides led to a substitution of alanine with asparagine at codon 593 (A593N) in the fifth transmembrane helix of TSHR. This somatic mutation resided on the same TSHR allele with the germline polymorphism D727E. The functional characteristics of the single TSHR mutants A593N and D727E and of the double mutant A593N/D727E were studied in transiently transfected COS-7 cells. The TSHR mutants A593N and A593N/D727E constitutively activated the cAMP cascade, whereas the D727E mutant did not differ from the wild-type TSHR. Surprisingly, the double mutant's specific constitutive activity was 2.3-fold lower than the A593N mutant. Thus, the polymorphism significantly ameliorates G(alphas) protein activation in the presence of the gain-of-function mutation A593N, although it is functionally inert in the context of the wild-type TSHR.     

Dihydrolipoamide dehydrogenase mutation alters the NADH sensitivity of pyruvate dehydrogenase complex of Escherichia coli K-12

Under anaerobic growth conditions, an active pyruvate dehydrogenase (PDH) is expected to create a redox imbalance in wild-type Escherichia coli due to increased production of NADH (>2 NADH molecules/glucose molecule) that could lead to growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetyl coenzyme A into reduced fermentation products, like alcohols, during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried an H322Y mutation (lpd102), while the other mutants carried an E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported anaerobic growth and homoethanol fermentation in an ldhA pflB double mutant. Enzyme kinetic studies revealed that the LPD(E354K) enzyme was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity of the appropriate PDH complex to NADH inhibition. The mutated forms of the PDH had a 10-fold-higher K(i) for NADH than the native PDH. The lower sensitivity of PDH to NADH inhibition apparently increased PDH activity in anaerobic E. coli cultures and created the new ethanologenic fermentation pathway in this bacterium. Analogous mutations in the LPD of other bacteria may also significantly influence the growth and physiology of the organisms in a similar fashion.     

Stabilization of the cold shock protein CspB from Bacillus subtilis by evolutionary optimization of Coulombic interactions

The bacterial cold shock proteins (Csp) are used by both experimentalists and theoreticians as model systems for analyzing the Coulombic contributions to protein stability. We employ Proside, a method of directed evolution, to identify stabilized variants of Bs-CspB from Bacillus subtilis. Proside links the increased protease resistance of stabilized protein variants to the infectivity of a filamentous phage. Here, three cspB libraries were used for in vitro selections to explore the stabilizing potential of charged amino acids in Bs-CspB. In the first library codons for nine selected surface residues were partially randomized, in the second one random mutations were introduced non-specifically by error-prone PCR, and in the third one the spontaneous mutation rate of the phage in Escherichia coli was used. Stabilizing mutations were found at the surface positions 1, 3, 46, 48, 65, and 66. The contributions of these mutations to stability were characterized by analyzing them individually and in combination. The best combination (M1R, E3K, K65I, and E66L) increased the midpoint of thermal unfolding of Bs-CspB from 53.8 to 85.0 degrees C. The effects of most mutations are strongly context dependent. A good example is provided by the E3R mutation. It is strongly stabilizing (DeltaDeltaGD=11.1kJ mol(-1)) in the wild-type protein, but destabilizing (DeltaDeltaGD=-4.0kJ mol(-1)) in the A46K/S48R/E66L variant. The stabilizations by charge mutations did not correlate well with the corresponding changes in the protein net charge, and they could not be ascribed to the formation of ion pairs. Previous theoretical analyses did not identify the stabilization caused by the mutations at positions 1, 46, and 48. Also, electrostatics calculations based on protein net charge or charge asymmetry did not predict well the stability changes that occur when charged residues in Bs-CspB are mutated. It remains a challenge to model the Coulombic interactions of charged residues in a protein and to determine their contributions to the Gibbs free energy of protein folding.     

Activating Mutations of the Serotonin 5-HT2C Receptor

Abstract: Site-directed mutagenesis was performed to create a mutant serotonin 5-HT_2C receptor that would mimic the active conformation of the native receptor. Structural alteration of receptor conformation was achieved by changing amino acid no. 312 from serine to phenylalanine (S312F) or lysine (S312K). After expression in COS-7 cells, the binding affinity of 5-HT for [³H]-mesulergine-labeled 5-HT_2C receptors increased from 203 n M (native) to 76 n M for S312F and 6.6 n M for S312K mutant receptors. 5-HT potency for stimulation of phosphatidylinositol (PI) hydrolysis increased from 70 n M (native) to 28 n M for S312F and 2.7 n M for S312K mutant receptors. The mutant receptors were constitutively active, stimulating PI hydrolysis in the absence of agonist. S312F and S312K mutations resulted in twofold and five-fold increases, respectively, in basal levels of PI hydrolysis. Mianserin and mesulergine displayed inverse agonist activity by decreasing basal levels of PI hydrolysis stimulated by S312K mutant receptors. [³H]5-HT and [³H]-mesulergine labeled the same number of S312K mutant receptors and 5'-guanylylimidodiphosphate had no effect on [³H]5-HT binding. These results indicate that serine → lysine mutation at amino acid no. 312 produces an agonist high-affinity state of the 5-HT_2C receptor that spontaneously couples to G proteins and stimulates PI hydrolysis in the absence of agonist.     

Crystal structure and molecular dynamics simulation of ubiquitin-like domain of murine parkin

Parkin is the gene product identified as the major cause of autosomal recessive juvenile Parkinsonism (AR-JP). Parkin, a ubiquitin ligase E3, contains a unique ubiquitin-like domain in its N-terminus designated Uld which is assumed to be a interaction domain with the Rpn 10 subunit of 26S proteasome. To elucidate the structural and functional role of Uld in parkin at the atomic level, the X-ray crystal structure of murine Uld was determined and a molecular dynamics simulation of wild Uld and its five mutants (K27N, R33Q, R42P, K48A and V56E) identified from AR-JP patients was performed. Murine Uld consists of two alpha helices [Ile23-Arg33 (alpha1) and Val56-Gln57 (alpha2)] and five beta strands [Met1-Phe7 (beta1), Tyr11-Asp18 (beta2), Leu41-Phe45 (beta3), Lys48-Pro51 (beta4) and Ser65-Arg72 (beta5)] and its overall structure is essentially the same as that of human ubiquitin with a 1.22 A rmsd for the backbone atoms of residues 1-76; however, the sequential identity and similarity between both molecules are 32% and 63%, respectively. This close resemblance is due to the core structure built by same hydrogen bond formations between and within the backbone chains of alpha1 and beta1/2/5 secondary structure elements and by nearly the same hydrophobic interactions formed between the nonpolar amino acids of their secondary structures. The side chain NetaH of Lys27 on the alpha1 helix was crucial to the stabilization of the spatial orientations of beta3 and beta4 strands, possible binding region with Rpn 10 subunit, through three hydrogen bonds. The MD simulations showed the K27N and R33Q mutations increase the structural fluctuation of these beta strands including the alpha1 helix. Reversely, the V56E mutant restricted the spatial flexibility at the periphery of the short alpha2 helix by the interactions between the polar atoms of Glu56 and Ser19 residues. However, a large fluctuation of beta4 strand with respect to beta5 strand was induced in the R42P mutant, because of the impossibility of forming paired hydrogen bonds of Pro for Arg42 in wild Uld. The X-ray structure showed that the side chains of Asp39, Gln40 and Arg42 at the N-terminal periphery of beta3 strand protrude from the molecular surface of Uld and participate in hydrogen bonds with the polar residues of neighboring Ulds. Thus, the MD simulation suggests that the mutation substitution of Pro for Arg42 not only causes the large fluctuation of beta3 strand in the Uld but also leads to the loss of the ability of Uld to trap the Rpn 10 subunit. In contrast, the MD simulation of K48A mutant showed little influence on the beta3-beta4 loop structure, but a large fluctuation of Lys48 side chain, suggesting the importance of flexibility of this side chain for the interaction with the Rpn 10 subunit. The present results would be important in elucidating the impaired proteasomal binding mechanism of parkin in AR-JP.     

Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast. Identification of point mutations that "silence" a constitutively active mutant M3 receptor and greatly impair receptor/G protein coupling

The M3 muscarinic receptor is a prototypical member of the class I family of G protein-coupled receptors (GPCRs). To facilitate studies on the structural mechanisms governing M3 receptor activation, we generated an M3 receptor-expressing yeast strain (Saccharomyces cerevisiae) that requires agonist-dependent M3 receptor activation for cell growth. By using receptor random mutagenesis followed by a genetic screen in yeast, we initially identified a point mutation at the cytoplasmic end of transmembrane domain (TM) VI (Q490L) that led to robust agonist-independent M3 receptor signaling in both yeast and mammalian cells. To explore further the molecular mechanisms by which point mutations can render GPCRs constitutively active, we subjected a region of the Q490L mutant M3 receptor that included TM V-VII to random mutagenesis. We then applied a yeast genetic screen to identify second-site mutations that could suppress the activating effects of the Q490L mutation and restore wild-type receptor-like function to the Q490L mutant receptor. This analysis led to the identification of 12 point mutations that allowed the Q490L mutant receptor to function in a fashion similar to the wild-type receptor. These amino acid substitutions mapped to two distinct regions of the M3 receptor, the exofacial segments of TM V and VI and the cytoplasmic ends of TM V-VII. Strikingly, in the absence of the activating Q490L mutation, all recovered point mutations severely reduced the efficiency of receptor/G protein coupling, indicating that the targeted residues play important roles in receptor activation and/or receptor/G protein coupling. This strategy should be generally applicable to identify sites in GPCRs that are critically involved in receptor function.     

Molecular dynamics simulation of the effects of single (S221P) and double (S221P and K216E) mutations in the hemagglutinin protein of influenza A H5N1 virus: a study on host receptor specificity

Avian influenza viruses of subtype H5N1 circulating in animals continue to pose threats to human health. The binding preference of the viral surface protein hemagglutinin (HA) to sialosaccharides of receptors is an important area for understanding mutations in the receptor binding site that could be the cause for avian-to-human transmission. In the present work, we studied the effect of two receptor binding site mutations, S221P singly and in combination with another mutation K216E in the HA protein of influenza A H5N1 viruses. Docking of sialic acid ligands corresponding to both avian and human receptors and molecular dynamics simulations of the complexes for wild and mutant strains of H5N1 viruses were carried out. The H5N1 strain possessing the S221P mutation indicated decreased binding to α2,3-linked sialic acids (avian receptor, SAα2,3Gal) when compared to the binding of the wild-type strain that did not possess the HA-221 mutation. The binding to α2,6-linked sialic acids (human receptor, SAα2,6Gal) was found to be comparable, indicating that the mutant strain shows limited dual receptor specificity. On the other hand, the S221P mutation in synergism with the K216E mutation in the binding site, resulted in increased binding affinity for SAα2,6Gal when compared to SAα2,3Gal, indicative of enhanced binding to human receptors. The in-depth study of the molecular interactions in the docked complexes could explain how co-occurring mutations in the HA viral protein can aid in providing fitness advantage to the virus, in the context of host receptor specificity in emerging variants of H5N1 influenza viruses.