Double mutation at the subunit interface of glutathione transferase rGSTM1-1 results in a stable, folded monomer

Canonical glutathione (GSH) transferases are dimeric proteins with subunits composed of an N-terminal GSH binding region (domain 1) and a C-terminal helical region (domain 2). The stabilities of several GSH transferase dimers are dependent upon two groups of interactions between domains 1 and 2 of opposing subunits: a hydrophobic ball-and-socket motif and a buried charge cluster motif. In rGSTM1-1, these motifs involve residues F56 and R81, respectively. The structural basis for the effects of mutating F56 to different residues on dimer stability and function has been reported (Codreanu et al. (2005) Biochemistry 44, 10605-10612). Here, we show that the simultaneous disruption of both motifs in the F56S/R81A mutant causes complete dissociation of the dimer to a monomeric protein on the basis of gel filtration chromatography and multiple-angle laser light scattering. The fluorescence and far-UV CD properties of the double mutant as well as the kinetics of amide H/D exchange along the polypeptide backbone suggest that the monomer has a globular structure that is similar to a single subunit in the native protein. However, the mutant monomer has severely impaired catalytic activity, suggesting that the dimer interface is vital for efficient catalysis. Backbone amide H/D exchange kinetics in the F56S and F56S/R81A mutants indicate that a reorganization of the loop structure between helix alpha2 and strand beta3 near the active site is responsible for the decreased catalytic activity of the monomer. In addition, the junction between the alpha4 and alpha5 helices in F56S/R81R shows decreased H/D exchange, indicating another structural change that may affect catalysis. Although the native subunit interface is important for dimer stability, urea-induced unfolding of the F56S/R81A mutant suggests that the interface is not essential for the thermodynamic stability of individual subunits. The H/D exchange data reveal a possible molecular basis for the folding cooperativity observed between domains 1 and 2.     

Influence of the dimer interface on glutathione transferase structure and dynamics revealed by amide H/D exchange mass spectrometry

Mammalian glutathione (GSH) transferases are dimeric proteins, many of which share a common hydrophobic interaction motif that is important for dimer stability. In the rGSTM1-1 enzyme this motif involves the side chain of F56, located on the 56 loop of the N-terminal domain, which is intercalated between the alpha4- and alpha5-helices of the C-terminal domain of the opposing subnuit. Disruption of the complementary interactions in this motif by mutation of F56 to serine, arginine, or glutamate is known to have deleterious effects on catalytic efficiency but remarkably different effects on the stability of the dimer [Hornby et al. (2002) Biochemistry 41, 14238-14247]. The structural basis for the behavior of the mutants by amide H/D exchange mass spectrometry is described. A substantial decrease in H/D exchange is observed in the GSH binding domain and in parts of the dimer interface upon substrate binding. The F56S and F56R mutants exhibit enhanced H/D exchange kinetics in the GSH binding domain and at the dimer interface. In contrast, the F56E mutant shows a decrease in the rate and extent of amide H/D exchange at the dimer interface and enhanced exchange kinetics in the GSH binding domain. The results suggest that the F56E mutant has a restructured dimer interface with decreased solvent accessibility and dynamics. Although all of the F56 mutations disrupt the GSH binding site, the effects of the mutations on the structure of the subunit interface and dimer stability are quite distinct.     

A conserved "hydrophobic staple motif" plays a crucial role in the refolding of human glutathione transferase P1-1

The specific (i, i+5) hydrophobic staple interaction involving a helix residue and a second residue located in the turn preceding the helix is a recurrent motif at the N terminus of alpha-helices. This motif is strictly conserved in the core of all soluble glutathione transferases (GSTs) as well as in other protein structures. Human GSTP1-1 variants mutated in amino acid Ile(149) and Tyr(154) of the hydrophobic staple motif of the alpha6-helix were analyzed. In particular, a double mutant cycle analysis has been performed to evaluate the role of the hydrophobic staple motif in the refolding process. The results show that this local interaction, by restricting the number of conformations of the alpha6-helix relative to the alpha1-helix, favors the formation of essential interdomain interactions and thereby accelerates the folding process. Thus, for the first time it is shown that the hydrophobic staple interaction has a role in the folding process of an intact protein. In P(i) class GSTs, Tyr(154) appears to be of particular structural importance, since it interacts with conserved residues Leu(21), Asp(24), and Gln(25) of the adjacent alpha1-helix which contributes to the active site. Human GSTP1-1 variants L21A and Y154F have also been analyzed in order to distinguish the role of interdomain interactions from that of the hydrophobic staple. The experimental results reported here suggest that the strict conservation of the hydrophobic staple motif reflects an evolutionary pressure for proteins to fold rapidly.     

Role of the C-terminal helix 9 in the stability and ligandin function of class alpha glutathione transferase A1-1

Helix 9 at the C-terminus of class alpha glutathione transferase (GST) polypeptides is a unique structural feature in the GST superfamily. It plays an important structural role in the catalytic cycle. Its contribution toward protein stability/folding as well as the binding of nonsubstrate ligands was investigated by protein engineering, conformational stability, enzyme activity, and ligand-binding methods. The helix9 sequence displays an unfavorable propensity toward helix formation, but tertiary interactions between the amphipathic helix and the GST seem to contribute sufficient stability to populate the helix on the surface of the protein. The helix's stability is enhanced further by the binding of ligands at the active site. The order of ligand-induced stabilization increases from H-site occupation, to G-site occupation, to the simultaneous occupation of H- and G-sites. Ligand-induced stabilization of helix9 reduces solvent accessible hydrophobic surface by facilitating firmer packing at the hydrophobic interface between helix and GST. This stabilized form exhibits enhanced affinity for the binding of nonsubstrate ligands to ligandin sites (i.e., noncatalytic binding sites). Although helix9 contributes very little toward the global stability of hGSTA1-1, its conformational dynamics have significant implications for the protein's equilibrium unfolding/refolding pathway and unfolding kinetics. Considering the high concentration of reduced glutathione in human cells (about 10 mM), the physiological form of hGSTA1-1 is most likely the thiol-complexed protein with a stabilized helix9. The C-terminus region (including helix9) of the class alpha polypeptide appears not to have been optimized for stability but rather for catalytic and ligandin function.     

Molecular basis for the structural instability of human DJ-1 induced by the L166P mutation associated with Parkinson's disease

DJ-1 is a dimeric protein of unknown function in vivo. A mutation in the human DJ-1 gene causing substitution of proline for leucine at residue 166 (L166P) has been linked to early onset Parkinson's disease. Lack of structural stability has precluded experimental determination of atomic-resolution structures of the L166P DJ-1 polymorph. We have performed multiple molecular dynamics (MD) simulations ( approximately 1/3 mus) of the wild-type and L166P DJ-1 polymorph at physiological temperature to predict specific structural effects of the L166P substitution. L166P disrupted helices alpha1, alpha5, alpha6 and alpha8 with alpha8 undergoing particularly severe disruption. Secondary structural elements critical for protein stability and dimerization were significantly disrupted across the entire dimer interface, as were extended hydrophobic surfaces involved in dimer formation. Relative to wild-type DJ-1, L166P DJ-1 populated a broader ensemble of structures, many of which corresponded to distorted conformations. In a L166P dimer model the substitution significantly destabilized the dimer interface, interrupting >100 intermolecular contacts that are important for dimer formation. The L166P substitution also led to major perturbations in the region of a highly conserved cysteine residue (Cys-106) that participates in dimerization and that is critical for a proposed chaperone function of DJ-1. Cys-106 is located approximately 16 A from the substitution site, demonstrating that structural disruptions propagate throughout the whole protein. Furthermore, L166P DJ-1 showed a significant increase in hydrophobic surface area relative to wild-type protein, possibly explaining the tendency of the mutant protein to aggregate. These simulations provide details about specific structural disturbances throughout L166P DJ-1 that previous studies have not revealed.     

Design of the helix-turn-helix motif: nonlocal effects of quaternary structure in DNA recognition investigated by laser Raman spectroscopy

The operator-binding domain of phage lambda repressor provides a model for DNA recognition by the helix-turn-helix (HTH) motif. In the wild-type protein, dimerization is mediated by hydrophobic packing (of the dyad-related helix 5), which serves as an indirect determinant of operator affinity. The mutant repressor, Tyr88----Cys, forms an intersubunit disulfide linkage and exhibits enhancement of both structural stability and operator affinity. Yet the dimer-specific operator affinity of the mutant is 10-fold weaker than that of the wild-type (noncovalent) dimer, suggesting nonlocal effects of the intersubunit disulfide bond on HTH recognition (Sauer et al., 1986). To explore such nonlocal effects, we describe laser Raman studies of the Cys88 mutant repressor and its interaction with operator sites OL1 and OR3. The following results have been obtained: (i) Wild-type and mutant dimers exhibit similar secondary structures, indicated by quantitative comparison of Raman amide I and amide III bands. (ii) The engineered disulfide of the mutant lacks rigorous symmetry; we observe mainly the gauche/gauche/trans CC-S-S-CC rotamer. (iii) Remarkably, distinctive local and nonlocal differences are observed in the mechanisms of DNA recognition by wild-type and mutant repressors. These differences involve specific hydrogen-bonding interactions between the protein and DNA, including guanine N7 sites in the major groove of DNA, and alterations in DNA phosphodiester conformation induced by protein binding. We analyze these differences in relation to crystal structures of the wild-type dimer with and without bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis in hemoglobin: functional and structural role of inter- and intrasubunit hydrogen bonds as studied with 37 beta and 145 beta mutations.

In order to clarify the functional and structural role of intra- and intersubunit hydrogen bonds in human hemoglobin (Hb A), we prepared two artificial beta chain mutant hemoglobins by site-directed mutagenesis. The mutant Hb Phe-37 beta, in which Trp-37 beta is replaced by Phe to remove the intersubunit hydrogen bond between Asp-94 alpha and Trp-37 beta at the alpha 1-beta 2 interface in deoxy Hb A, showed a markedly increased oxygen affinity and almost completely diminished Bohr effect and cooperativity. However, 1H-NMR data indicated that the structure of deoxy Hb Phe-37 beta is rather similar to that of deoxy Hb A. The enhanced tetramer-to-dimer dissociation previously observed in Hb Hirose (Trp-37 beta----Ser) together with our observation of the effects of organic phosphate on the structure and function of Hb Phe-37 beta suggested that a large part of the abnormal properties of Hb Phe-37 beta observed for dilute solutions appears to result from partial dissociation into alpha beta dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated state. Thus, the primary and direct role of the hydrogen bond between Asp-94 alpha and Trp-37 beta is to stabilize the tetrameric assembly, and thereby this hydrogen bond indirectly contributes to stabilization of the T-quaternary structure. The other mutant Hb Phe-145 beta has a Phe residue at the 145 beta site and lacks the intrasubunit hydrogen bond formed between Tyr-145 beta and the carbonyl group of Val-98 beta in deoxy Hb A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variable contributions of tyrosine residues to the structural and spectroscopic properties of the factor for inversion stimulation

The diverse roles of tyrosine residues in proteins may be attributed to their dual hydrophobic and polar nature, which can result in hydrophobic and ring stacking interactions, as well as hydrogen bonding. The small homodimeric DNA binding protein, factor for inversion stimulation (FIS), contains four tyrosine residues located at positions 38, 51, 69, and 95, each involved in specific intra- or intermolecular interactions. To investigate their contributions to the stability, flexibility, and spectroscopic properties of FIS, each one was independently mutated to phenylalanine. Equilibrium denaturation experiments show that Tyr95 and Tyr51 stabilize FIS by about 2 and 1 kcal/mol, respectively, as a result of their involvement in a hydrogen bond-salt bridge network. In contrast, Tyr38 destabilizes FIS by about 1 kcal/mol due to the placement of a hydroxyl group in a hydrophobic environment. The stability of FIS was not altered when the solvent-exposed Tyr69 was mutated. Limited proteolysis with trypsin and V8 proteases was used to monitor the flexibility of the C-terminus (residues 71-98) and the dimer core (residues 26-70), respectively. The results for Y95F and Y51F FIS revealed a different proteolytic susceptibility of the dimer core compared to the C-terminus, suggesting an increased flexibility of the latter. DNA binding affinity of the various FIS mutants was only modestly affected and correlated inversely with the C-terminal flexibility probed by trypsin proteolysis. Deconvolution of the fluorescence contribution of each mutant revealed that it varies in intensity and direction for each tyrosine in WT FIS, highlighting the role of specific interactions and the local environment in determining the fluorescence of tyrosine residues. The significant changes in stability, flexibility, and signals observed for the Y51F and Y95F mutations are attributed to their coupled participation in the hydrogen bond-salt bridge network. These results highlight the importance of tyrosine hydrogen-bonding and packing interactions for the stability of FIS and demonstrate the varying roles that tyrosine residues can play on the structural and spectroscopic properties of even small proteins.     

Crystal structure and mutational analysis of the Saccharomyces cerevisiae cell cycle regulatory protein Cks1: implications for domain swapping, anion binding and protein interactions

BACKGROUND: The Saccharomyces cerevisiae protein Cks1 (cyclin-dependent kinase subunit 1) is essential for cell-cycle progression. The biological function of Cks1 can be modulated by a switch between two distinct molecular assemblies: the single domain fold, which results from the closing of a beta-hinge motif, and the intersubunit beta-strand interchanged dimer, which arises from the opening of the beta-hinge motif. The crystal structure of a cyclin-dependent kinase (Cdk) in complex with the human Cks homolog CksHs1 single-domain fold revealed the importance of conserved hydrophobic residues and charged residues within the beta-hinge motif. RESULTS: The 3.0 A resolution Cks1 structure reveals the strict structural conservation of the Cks alpha/beta-core fold and the beta-hinge motif. The beta hinge identified in the Cks1 structure includes a novel pivot and exposes a cluster of conserved tyrosine residues that are involved in Cdk binding but are sequestered in the beta-interchanged Cks homolog suc1 dimer structure. This Cks1 structure confirms the conservation of the Cks anion-binding site, which interacts with sidechain residues from the C-terminal alpha helix of another subunit in the crystal. CONCLUSIONS: The Cks1 structure exemplifies the conservation of the beta-interchanged dimer and the anion-binding site in evolutionarily distant yeast and human Cks homologs. Mutational analyses including in vivo rescue of CKS1 disruption support the dual functional roles of the beta-hinge residue Glu94, which participates in Cdk binding, and of the anion-binding pocket that is located 22 A away and on an opposite face to Glu94. The Cks1 structure suggests a biological role for the beta-interchanged dimer and the anion-binding site in targeting Cdks to specific phosphoproteins during cell-cycle progression.     

The mechanism of aubstrate eecognition of pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as determined by X-ray crystallography and site-directed mutagenesis

Pyroglutamyl-peptidase is able to specifically remove the amino-terminal pyroglutamyl residue protecting proteins or peptides from aminopeptidases. To clarify the mechanism of substrate recognition for the unique structure of the pyrrolidone ring, x-ray crystallography and site-directed mutagenesis were applied. The crystal structure of pyroglutamyl-peptidase bound to a transition state analog inhibitor (Inh), pyroglutaminal, was determined. Two hydrogen bonds were located between the main chain of the enzyme and the inhibitor (71:O.H-N:Inh and Gln71:N-H.OE:Inh), and the pyrrolidone ring of the inhibitor was inserted into the hydrophobic pocket composed of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143. To study in detail the hydrophobic pocket, Phe-10, Phe-13, and Phe-142 were selected for mutation experiments. The k(cat) value of the F10Y mutant decreased, but the two phenylalanine mutants F13Y and F142Y did not exhibit significant changes in kinetic parameters compared with the wild-type enzyme. The catalytic efficiencies (k(cat)/K(m)) for the F13A and F142A mutants were less than 1000-fold that of the wild-type enzyme. The x-ray crystallographic study of the F142A mutant showed no significant change except for a minor one in the hydrophobic pocket compared with the wild type. These findings indicate that the molecular recognition of pyroglutamic acid is achieved through two hydrogen bonds and an insertion in the hydrophobic pocket. In the pocket, Phe-10 is more important to the hydrophobic interaction than is Phe-142, and furthermore Phe-13 serves as an "induced fit" mechanism.     

Novel aromatic residues in transmembrane domains IV and V involved in agonist binding at alpha(1a)-adrenergic receptors

We examined the role that aromatic residues located in the transmembrane helices of the alpha(1a)-adrenergic receptor play in promoting antagonist binding. Since alpha(1)-antagonists display low affinity binding at beta(2)-adrenergic receptors, two phenylalanine residues, Phe-163 and Phe-187, of the alpha(1a)-AR were mutated to the corresponding beta(2)-residue. Neither F163Q nor F187A mutations of the alpha(1a) had any effect on the affinity of the alpha(1)-antagonists. However, the affinity of the endogenous agonist epinephrine was reduced 12.5- and 8-fold by the F163Q and F187A mutations, respectively. An additive loss in affinity (150-fold) for epinephrine was observed at an alpha(1a) containing both mutations. The loss of agonist affinity scenario could be reversed by a gain of affinity with mutation of the corresponding residues in the beta(2) to the phenylalanine residues in the alpha(1a). We propose that both Phe-163 and Phe-187 are involved in independent aromatic interactions with the catechol ring of agonists. The potency but not the efficacy of epinephrine in stimulating phosphatidylinositol hydrolysis was reduced 35-fold at the F163Q/F187A alpha(1a) relative to the wild type receptor. Therefore, Phe-163 and Phe-187 represent novel binding contacts in the agonist binding pocket of the alpha(1a)-AR, but are not involved directly in receptor activation.     

F99 is Critical for Dimerization and Activation of South African HIV-1 Subtype C Protease

HIV-1 protease (PR) is an obligate homodimer which plays a pivotal role in the maturation and hence propagation of HIV. Although successful developments on PR active site inhibitors have been achieved, the major limiting factor has been the emergence of HIV drug-resistant strains. Disruption of the dimer interface serves as an alternative mechanism to inactivate the enzyme. The terminal residue, F99, was mutated to an alanine to investigate its contribution to dimer stability in the South African HIV-1 subtype C (C-SA) PR. The F99A PR and wild-type C-SA PR were overexpressed and purified. The activities of the PRs and their ability to bind an active site inhibitor, acetyl-pepstatin, were determined in vitro. The F99A PR showed no activity and the inability to bind to the inhibitor. Secondary and quaternary structure analysis were performed and revealed that the F99A PR is monomeric with reduced β-sheet content. The mutation of F99 to alanine disrupted the presumed ‘lock-and-key’ motif at the terminal dimer interface, in turn creating a cavity at the N- and C-terminal antiparallel β-sheet. These findings support the design of inhibitors targeting the C-terminus of the C-SA PR, centered on interactions with the bulky F99.     

Molecular recognition at the dimer interface of a class mu glutathione transferase: role of a hydrophobic interaction motif in dimer stability and protein function

Cytosolic glutathione (GSH) transferases (GSTs) exist as stable homo- and heterodimers. Interactions at the subunit interface serve an important role in stabilizing the subunit tertiary structures of all GSH transferases. In addition, the dimer is required to maintain functional conformations at the active site on each subunit and the nonsubstrate ligand binding site at the dimer interface [Dirr, H. W. (2001) Chem.-Biol. Interact. 133, 19-23]. In this study, we report on the contribution of a specific intersubunit hydrophobic motif in rGSTM1-1 to dimer stability and protein function. The motif consists of the side chain of F56 from one subunit intercalated between helices 4 and 5 of the second subunit. Replacement of F56 with the hydrophilic side chains of serine, arginine, and glutamate results in a change in the structure of the active site, a marked diminution in catalytic efficiency, and alterations in the ability to bind nonsubstrate ligands. The mutations also affect the ability of the enzyme to bind GSH and the substrate analogue glutathione sulfonate. The functionality of rGSTM1-1 was disrupted to the greatest extent for the F56E mutant. Though mutations at this position do not alter the three-state equilibrium folding process for rGSTM1-1 (i.e., N(2) <--> 2I <--> 2U), destabilizing mutations at position 56 shift the equilibrium between the folded dimer (N(2)) and the monomeric intermediate (I) toward the latter conformational state. The transition to the unfolded state (U) is not significantly affected. The folded monomeric intermediate is also observed by electrospray ionization mass spectrometry. The amount of the intermediate is dependent on protein concentration and the residue at position 56. Mutations at position 56 have little impact on the secondary structure and stability of the monomeric folding intermediate. The dimerization process is proposed to induce a conformational change in the loop containing F56, resulting in improved stability and increased affinity between the M1 subunits.     

Simulation of the N-terminus of HIV-1 glycoprotein 41000 fusion peptide in micelles.

In this paper, the N-terminus of glycoprotein-41, the HIV-1 fusion peptide, was studied by molecular dynamics simulations in an explicit sodium dodecyl sulfate micelle. The simulation provides a detailed picture of the equilibrium structure and peptide stability as it interacts with the micelle. The equilibrium location of the peptide shows the peptide at the surface of the micelle with hydrophobic residues interacting with the micelle's core. At equilibrium, the peptide adopts an alpha-helical structure from residues 5-16 and a type-1 beta-turn from 17-20 with the other residues exhibiting more flexible conformations. The primary hydrophobic interactions with the micelle are from the leucine and phenylalanine residues (Leu-7, Phe-8, Leu-9, Phe-11, Leu-12) while the alanine and glycine residues (Ala-1, Gly-3, Gly-5, Ala-6, Gly-10, Gly-13, Ala-14, Ala-15, Gly-16, Gly-10, Ala-21) interact favorably with water molecules. The results suggest that Phe-8, part of the highly conserved FLG motif of the fusion peptide, plays a key role in the interaction of the peptide with membranes. Our simulations corroborate experimental investigations of the fusion peptide in SDS micelles, providing a high-resolution picture that explains the experimental findings.     

Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF-2 alpha kinase DAI in Saccharomyces cerevisiae.

The protein kinase DAI is activated upon viral infection of mammalian cells and inhibits protein synthesis by phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2 alpha). DAI is activated in vitro by double-stranded RNAs (dsRNAs), and binding of dsRNA is dependent on two copies of a conserved sequence motif located N terminal to the kinase domain in DAI. High-level expression of DAI in Saccharomyces cerevisiae cells is lethal because of hyperphosphorylation of eIF-2 alpha; at lower levels, DAI can functionally replace the protein kinase GCN2 and stimulate translation of GCN4 mRNA. These two phenotypes were used to characterize structural requirements for DAI function in vivo, by examining the effects of amino acid substitutions at matching positions in the two dsRNA-binding motifs and of replacing one copy of the motif with the other. We found that both copies of the dsRNA-binding motif are required for high-level kinase function and that the N-terminal copy is more important than the C-terminal copy for activation of DAI in S. cerevisiae. On the basis of these findings, we conclude that the requirements for dsRNA binding in vitro and for activation of DAI kinase function in vivo closely coincide. Two mutant alleles containing deletions of the first or second binding motif functionally complemented when coexpressed in yeast cells, strongly suggesting that the active form of DAI is a dimer. In accord with this conclusion, overexpression of four catalytically inactive alleles containing different deletions in the protein kinase domain interfered with wild-type DAI produced in the same cells. Interestingly, three inactivating point mutations in the kinase domain were all recessive, suggesting that dominant interference involves the formation of defective heterodimers rather than sequestration of dsRNA activators by mutant enzymes. We suggest that large structural alterations in the kinase domain impair an interaction between the two protomers in a DAI dimer that is necessary for activation by dsRNA or for catalysis of eIF-2 alpha phosphorylation.     

Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.     

Edge-to-face CH/pi interaction between ligand Phe-phenyl and receptor aromatic group in the thrombin receptor activation

In the ligand/receptor interaction, the side chain phenyl group of phenylalanine (Phe) is involved in a so-called hydrophobic interaction, in which the Phe-phenyl group functions as a p element or merely as a hydrophobic element. The thrombin receptor-tethered ligand SFLLRNP consists of the Phe-2 residue essential for receptor activation. In order to explore the molecular characteristics of this Phe-2-phenyl group, a complete set of S/Phe/LLRNP peptides comprising six different difluorophenylalanine isomers [(F(2))Phe] was newly synthesized and assayed to evaluate their ability to induce the aggregation of human platelets. The assay results clarified several important structural elements to conclude that Phe-2-phenyl of S/Phe/LLRNP is in the edge-to-face CH/pi interaction with the receptor aromatic group, utilizing the Phe-phenyl edge along with adjacent benzene hydrogens at positions (2-3) or (5-6). It was also found that the fluorine atom at position 4 increases the acidity of the hydrogen mainly at its ortho position, resulting in a reinforcement of the CH/pi interaction and thus in an enhancement of biological activity. The H-->F replacement in the benzene ring was found to provide an effective structural examination to the Phe residue; i.e., to identify the hydrogens in the CH/pi interaction, and to strengthen the CH/pi interaction.     

Analysis of the FLVR motif of SHIP1 and its importance for the protein stability of SH2 containing signaling proteins

Binding of proteins with SH2 domains to tyrosine-phosphorylated signaling proteins is a key mechanism for transmission of biological signals within the cell. Characterization of dysregulated proteins in cell signaling pathways is important for the development of therapeutic approaches. The AKT pathway is a frequently upregulated pathway in most cancer cells and the SH2-containing inositol 5-phosphatase SHIP1 is a negative regulator of the AKT pathway. In this study we investigated different mutations of the conserved FLVR motif of the SH2 domain and putative phosphorylation sites of SHIP1 which are located in close proximity to its FLVR motif. We demonstrate that patient-derived SHIP1-FLVR motif mutations e.g. F28L, and L29F possess reduced protein expression and increased phospho-AKT-S473 levels in comparison to SHIP1 wildtype. The estimated half-life of SHIP1-F28L protein was reduced from 23.2 h to 0.89 h in TF-1 cells and from 4.7 h to 0.6 h in Jurkat cells. These data indicate that the phenylalanine residue at position 28 of SHIP1 is important for its stability. Replacement of F28 with other aromatic residues like tyrosine and tryptophan preserves protein stability while replacement with non-aromatic amino acids like leucine, isoleucine, valine or alanine severely affects the stability of SHIP1. In consequence, a SHIP1-mutant with an aromatic amino acid at position 28 i.e. F28W can rescue the inhibitory function of wild type SHIP1, whereas SHIP1-mutants with non-aromatic amino acids i.e. F28V do not inhibit cell growth anymore. A detailed structural analysis revealed that F28 forms hydrophobic surface contacts in particular with W5, I83, L97 and P100 which can be maintained by tyrosine and tryptophan residues, but not by non-aromatic residues at position 28. In line with this model of mutation-induced instability of SHIP1-F28L, treatment of cells with proteasomal inhibitor MG132 was able to rescue expression of SHIP1-F28L. In addition, mutation of putative phosphorylation sites S27 and S33 adjacent to the FLVR motif of SHIP1 have an influence on its protein stability. These results further support a functional role of SHIP1 as tumor suppressor protein and indicate a regulation of protein expression of SH2 domain containing proteins via the FLVR motif.     

Effects of high pressure and temperature on the wild-type and F29W mutant forms of the N-domain of avian troponin C

The N-domain of troponin C (residues 1-90) regulates muscle contraction through conformational changes induced by Ca2+ binding. A mutant form of the isolated domain of avian troponin C (F29W) has been used in previous studies to observe conformational changes that occur upon Ca2+ binding, and pressure and temperature changes. Here we set out to determine whether the point mutation itself has any effects on the protein structure and its stability to pressure and temperature in the absence of Ca2+. Molecular dynamics simulations of the wild-type and mutant protein structures suggested that both structures are identical except in the main chain and the loop I region near the mutation site. Also, the simulations proposed that an additional cavity had been created in the core of the mutant protein. To determine whether such a cavity would affect the behavior of the protein when subjected to high pressures and temperatures, we performed 1H-NMR experiments at 300, 400, and 500 MHz on the wild-type and F29W mutant forms of the chicken N-domain troponin C in the absence of Ca2+. We found that the mutant protein at 5 kbar pressures had a destabilized beta-sheet between the Ca2+-binding loops, an altered environment near Phe-26, and reduced local motions of Phe-26 and Phe-75 in the core of the protein, probably due to a higher compressibility of the mutant. Under the same pressure conditions, the wild-type domain exhibited little change. Furthermore, the hydrophobic core of the mutant protein denatured at temperatures above 47 degrees C, while the wild-type was resistant to denaturation up to 56 degrees C. This suggests that the partially exposed surface mutation (F29W) significantly destabilizes the N-domain of troponin C by altering the packing and dynamics of the hydrophobic core.