Essential Arginine Residue Research Articles

GAP positions catalytic h-Ras residue Q61 for GTP hydrolysis in MD simulations

Ras binds to a GTPase activating protein (GAP) to turn off the cell propagation signal. Together, they form a transition state that has a very precise configuration that catalyzes GTP hydrolysis. GAP provides an essential arginine residue to the transition state. Fluorescence experiments show that an arginine analogue alone cannot restore Ras function. We hypothesize that the shared interface between the proteins plays a critical role in the Ras-GAP transition state. We perform molecular dynamics simulations of the Ras-GAP complex and of Ras to do a comparison of Ras structural features and conformational freedom.

PAMPs, DAMPs and SAMPs: Host Glycans are Self‐Associated Molecular Patterns, but subject to Microbial Molecular Mimicry

It has long been known that Pathogen-Associated Molecular Patterns (PAMPs, e.g., lipo-oligosaccharides, LOS) and Danger-Associated Molecular Patterns (DAMPs, e.g., fragments of endogenous hyaluronan) can be glycans or modified glycans. We have more recently emphasized that host glycans can also serve as Self-Associated Molecular Patterns (SAMPs), e.g., sialic acid-bearing glycans that are recognized by CD33-related Siglecs on innate immune cells to dampen responses, or by complement Factor H to dampen complement-mediated activation. However, several commensal or pathogenic organisms have evolved successful molecular mimicry of sialylated SAMPs, so as to hijack these mechanisms and suppress innate immune responses against them. Paired activating CD33rSiglecs have emerged in some lineages, presumably to counter hijacking by microbial molecular mimicry of sialylated SAMPs. Meanwhile, sialylated host SAMPs also have to evolve rapidly to escape from other pathogens that utilize them as targets to initiate infection. The ongoing pressures of these often-competing selection forces can explain our finding that the sialic-acid binding domains of CD33rSiglecs and Factor H show much higher than expected Ka/Ks ratios (an excess of non-synonymous substitutions changing encoded amino acids, over synonymous substitutions). Examples can also be found of lineage-specific complete loss of sialic binding e.g., via mutation of an essential arginine residue in some CD33rSiglec binding pocket––or even whole-sale elimination of the binding domain e.g., alternate splicing out of the amino-terminal V-set domain of CD33/Siglec-3. Such evolutionary changes can be either fixed or polymorphic within a given species. Further complexity arises because CD33rSiglec and Factor H can be directly engaged via protein:protein interactions, both with endogenous proteins such as Hsp70 and Vimentin, and with surface proteins of commensals e.g., the PorB protein of N. gonorrhea. The overall outcome is inter-species or intra-species variations in the intensity of innate immune responses, which can be reflected in relative susceptibility or resistance to infectious agents and/or harmful aggravation of endogenous inflammatory diseases. Perhaps because of the major changes in the sialome resulting from inactivation of the CMAH gene in the Homo lineage leading to humans, these and many other types of evolutionary changes appear to be very prominent in our species and in our pathogen regimes. This presentation will provide an overview of these topics and examples of our recent work in these areas. Varki, A.: Since there are PAMPs and DAMPs, there must be SAMPs? Glycan “self-associated molecular patterns” dampen innate immunity, but pathogens can mimic them. Glycobiology, 21:1121-1124, 2011. Padler-Karavani, V., Hurtado-Ziola, N., Chang, Y.-C., Sonnenburg, J., Ronaghy, A., Yu, H., Verhagen, A., Nizet, V., Chen, X., Varki, N., Varki, A., Angata, T.: Rapid Evolution of Binding Specificities and Expression Patterns of Inhibitory CD33-related Siglecs in Primates. FASEB J. 28:1280-93, 2014. Laubli, H. and Varki, A. Sialic acid–binding immunoglobulin-like lectins (Siglecs) detect self-associated molecular patterns to regulate immune responses. Cell. Mol. Life Sci. Springer, International. 2019.

Structural basis for non-radical catalysis by TsrM, a radical SAM methylase.

TsrM methylates C2 of the indole ring of L-tryptophan (Trp) during the biosynthesis of the quinaldic acid moiety of thiostrepton. It is annotated as a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase; however, TsrM does not reductively cleave SAM to the universal 5ʹ-deoxyadenosyl 5ʹ-radical intermediate, a hallmark of radical-SAM (RS) enzymes. Herein, we report structures of TsrM from Kitasatospora setae, the first of a cobalamin-dependent radical SAM methylase. Unexpectedly, the structures show an essential arginine residue that resides in the proximal coordination sphere of the cobalamin cofactor and a [4Fe–4S] cluster that is ligated by a glutamyl residue and three cysteines in a canonical CxxxCxxC RS motif. Structures in the presence of substrates suggest a substrate-assisted mechanism of catalysis, wherein the carboxylate group of SAM serves as a general base to deprotonate N1 of the tryptophan substrate, facilitating formation of a C2 carbanion.

PAMPs, DAMPs and SAMPs: Host Glycans are Self‐Associated Molecular Patterns, but subject to Microbial Molecular Mimicry

It has long been known that Pathogen‐Associated Molecular Patterns (PAMPs, e.g., lipooligosaccharides, LOS) and Danger‐Associated Molecular Patterns (DAMPs, e.g., fragments of endogenous hyaluronan) can be glycans or modified glycans. We have more recently emphasized that host glycans can also serve as Self‐Associated Molecular Patterns (SAMPs), e.g., sialic acid‐bearing glycans that are recognized by CD33‐related Siglecs on innate immune cells to dampen responses, or by complement Factor H to dampen complement‐mediated activation. However, several commensal or pathogenic organisms have evolved successful molecular mimicry of sialylated SAMPs, so as to hijack these mechanisms and suppress innate immune responses against them. Paired activating CD33rSiglecs have emerged in some lineages, presumably to counter hijacking by microbial molecular mimicry of sialylated SAMPs. Meanwhile, sialylated host SAMPs also have to evolve rapidly to escape from other pathogens that utilize them as targets to initiate infection. The ongoing pressures of these often‐competing selection forces can explain our finding that the sialic‐acid binding domains of CD33rSiglecs and Factor H show much higher than expected Ka/Ks ratios (an excess of non‐synonymous substitutions changing encoded amino acids, over synonymous substitutions). Examples can also be found of lineage‐specific complete loss of sialic binding e.g., via mutation of an essential arginine residue in some CD33rSiglec binding pocket—or even whole‐sale elimination of the binding domain e.g., alternate splicing out of the amino‐terminal V‐set domain of CD33/Siglec‐3. Such evolutionary changes can be either fixed or polymorphic within a given species. Further complexity arises because CD33rSiglec and Factor H can be directly engaged via protein:protein interactions, both with endogenous proteins such as Hsp70 and Vimentin, and with surface proteins of commensals e.g., the PorB protein of N. gonorrhea. The overall outcome is inter‐species or intra‐species variations in the intensity of innate immune responses, which can be reflected in relative susceptibility or resistance to infectious agents and/or harmful aggravation of endogenous inflammatory diseases. Perhaps because of the major changes in the sialome resulting from inactivation of the CMAH gene in the Homo lineage leading to humans, these and many other types of evolutionary changes appear to be very prominent in our species and in our pathogen regimes. This presentation will provide an overview of these topics and examples of our recent work in these areas.

Function of essential chloride and arginine residue in nucleotide binding to vesicular nucleotide transporter

Vesicular nucleotide transporter (VNUT) plays a key role in purinergic signalling through its ability to transport nucleotides. VNUT belongs to the SLC17 family, which includes vesicular glutamate transporters (VGLUTs) and Type I Na+/phosphate cotransporters. All of these transporters exhibit membrane potential and Cl--dependent organic anion transport activity and have essential arginine in the transmembrane region. Previously, we reported that ketoacids inhibit these transporters through modulation of Cl- activation. Although this regulation is important to control signal transmission, the mechanisms underlying Cl--dependent regulation are unclear. Here, we examined the functional roles of Cl- and essential arginine residue on ATP binding to VNUT using the fluorescent ATP analogue trinitrophenyl-ATP (TNP-ATP). The fluorescence of TNP-ATP was enhanced by VNUT, whereas no enhancement was observed by VGLUT. Concentration-dependence curves showed that TNP-ATP was a high-affinity fluorescent probe for VNUT, with a Kd of 4.8 μM. TNP-ATP binding was competitive to ATP and showed similar specificity to transport activity. Addition of Cl- and ketoacids did not affect the apparent affinity for TNP-ATP. The Arg119 to Ala mutant retained TNP-ATP binding ability with slightly reduced affinity. Overall, these results indicated that Cl- and essential arginine were not important for ATP binding.

Folding of Gα Subunits: Implications for Disease States.

G-proteins play a central role in signal transduction by fluctuating between “on” and “off” phases that are determined by a conformational change. cAMP is a secondary messenger whose formation is inhibited or stimulated by activated Giα1 or Gsα subunit. We used tryptophan fluorescence, UV/vis spectrophotometry, and circular dichroism to probe distinct structural features within active and inactive conformations from wild-type and tryptophan mutants of Giα1 and Gsα. For all proteins studied, we found that the active conformations were more stable than the inactive conformations, and upon refolding from higher temperatures, activated wild-type subunits recovered significantly more native structure. We also observed that the wild-type subunits partially regained the ability to bind nucleotide. The increased compactness observed upon activation was consistent with the calculated decrease in solvent accessible surface area for wild-type Giα1. We found that as the temperature increased, Gα subunits, which are known to be rich in α-helices, converted to proteins with increased content of β-sheets and random coil. For active conformations from wild-type and tryptophan mutants of Giα1, melting temperatures indicated that denaturation starts around hydrophobic tryptophan microenvironments and then radiates toward tyrosine residues at the surface, followed by alteration of the secondary structure. For Gsα, however, disruption of secondary structure preceded unfolding around tyrosine residues. In the active conformations, a π-cation interaction between essential arginine and tryptophan residues, which was characterized by a fluorescence-measured red shift and modeled by molecular dynamics, was also shown to be a contributor to the stability of Gα subunits. The folding properties of Gα subunits reported here are discussed in the context of diseases associated to G-proteins.

Mechanism of 53BP1 activity regulation by RNA-binding TIRR and a designer protein.

Dynamic protein interaction networks such as DNA double-strand break (DSB) signaling are modulated by post-translational modifications. The DNA repair factor 53BP1 is a rare example of a protein whose post-translational modification-binding function can be switched on and off. 53BP1 is recruited to DSBs by recognizing histone lysine methylation within chromatin, an activity directly inhibited by the 53BP1-binding protein TIRR. X-ray crystal structures of TIRR and a designer protein bound to 53BP1 now reveal a unique regulatory mechanism in which an intricate binding area centered on an essential TIRR arginine residue blocks the methylated-chromatin-binding surface of 53BP1. A 53BP1 separation-of-function mutation that abolishes TIRR-mediated regulation in cells renders 53BP1 hyperactive in response to DSBs, highlighting the key inhibitory function of TIRR. This 53BP1 inhibition is relieved by TIRR-interacting RNA molecules, providing proof-of-principle of RNA-triggered 53BP1 recruitment to DSBs.

ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter

Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment.

Trans-Acting Arginine Residues in the AAA+ Chaperone ClpB Allosterically Regulate the Activity through Inter- and Intradomain Communication

The molecular chaperone ClpB/Hsp104, a member of the AAA+ superfamily (ATPases associated with various cellular activities), rescues proteins from the aggregated state in collaboration with the DnaK/Hsp70 chaperone system. ClpB/Hsp104 forms a hexameric, ring-shaped complex that functions as a tightly regulated, ATP-powered molecular disaggregation machine. Highly conserved and essential arginine residues, often called arginine fingers, are located at the subunit interfaces of the complex, which also harbor the catalytic sites. Several AAA+ proteins, including ClpB/Hsp104, possess a pair of such trans-acting arginines in the N-terminal nucleotide binding domain (NBD1), both of which were shown to be crucial for oligomerization and ATPase activity. Here, we present a mechanistic study elucidating the role of this conserved arginine pair. First, we found that the arginines couple nucleotide binding to oligomerization of NBD1, which is essential for the activity. Next, we designed a set of covalently linked, dimeric ClpB NBD1 variants, carrying single subunits deficient in either ATP binding or hydrolysis, to study allosteric regulation and intersubunit communication. Using this well defined environment of site-specifically modified, cross-linked AAA+ domains, we found that the conserved arginine pair mediates the cooperativity of ATP binding and hydrolysis in an allosteric fashion.

Abstract 3955: 53BP1 facilitates the ATM-dependent phosphorylation of APLF in the DNA damage response

Abstract Aprataxin and polynucleotide kinase-like factor (APLF) is a DNA repair factor which facilitates DNA repair in part through interactions with its forkhead-associated (FHA) domain. The APLF FHA domain mediates interactions with specific threonine phosphorylated epitopes via a functionally conserved and essential arginine residue (Arg-27). In the context of DNA double-strand break (DSB) repair, substitution of APLF amino acid residue Arg-27 to alanine abolishes interactions with the DSB repair scaffold protein XRCC4, and impairs DSB repair. APLF is also a component of the ATM-dependent DNA damage response (DDR), which also includes 53BP1, and undergoes ionizing-radiation induced phosphorylation at Ser-116 facilitating its role in DSB repair. Here we demonstrate that the APLF FHA domain is essential for Ser-116 phosphorylation and for the association with 53BP1, which collectively enhance the accumulation of APLF at sites of DNA damage. The level of 53BP1 expression, but not XRCC4, was found to correlate with the phosphorylation of APLF by ATM, and disruption of the APLF-53BP1 interaction was associated with impaired cell survival following DNA damage. These results suggest that the APLF FHA domain directs interactions with multiple partners in the DNA damage response, and that 53BP1 facilitates the ATM-dependent phosphorylation of APLF. Citation Format: Amanda L. Fenton, Diana Tran, Christine Anne Koch. 53BP1 facilitates the ATM-dependent phosphorylation of APLF in the DNA damage response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3955. doi:10.1158/1538-7445.AM2014-3955

Structure basis of Leukotriene C4 Synthase and its isophtalate inhibitors

Leukotriene (LT) C4 synthase (LTC4S) is a key enzyme for the production of cysteinyl leukotrienes, LTC4, LTD4 and LTE4, which are relevance to asthma and allergy. LTC4S catalyzes the conjugation of glutathione (GSH) to LTA4. The crystal structure of LTC4S complex with GSH revealed the active sites locate at the interfaces of adjacent monomer in trans-membrane homo-trimer [1]. The unique U-shaped GSH binds the inner hydrophilic interface cavity and the amphiphilic LTA4 was proposed to bind in the hydrophobic V-shaped crevasse on the interface of the trimer hydrophobic surface. Two essential arginine residues was proved to exert as acid-base catalysis at the both sides of two substrates in highly regio- and stereo-selective manner enzymatically and crystallographically [2]. The architecture of the catalysis with Arg104 and Arg31 residues is the unique among various Glutathione-S-transferases. Arg104 activates and stabilizes the thiorate anaion of the bound GSH and Arg31 stabilize and activate epoxy of the other substrate LTA4, and each mutation causes not only reduction of activity but also each substrate binding affinity substantially. Furthermore, the putative LTA4 binding position was confirmed using the anomalous signal of selenium of the bound seleno-dodecylmaltoside (Se-DDM) at the V-shaped crevasse. To identify leads for novel therapeutics, we attempted to search competitive inhibitors against the unique shaped GSH binding site to discriminate the GSH binding sites of other GSTs that accommodate only its extending backbone conformer [3]. Hierarchical in silico screenings of 6 million compounds provided 300,000 dataset for docking, and after energy minimization based on the crystal structure of LTC4S, 111 compounds were selected as candidates for a competitive inhibitor to glutathione. 5-(5-Methylene-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid moiety was identified to inhibit LTC4 formation both an enzyme assay and a whole-cell assay. Finally, 5-((Z)-5-((E)-2-methyl-3-phenylallylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid was found to be the most potent inhibitor with 1.9 µM of IC50, and in the whole-cell assay to inhibit LTC4 synthesis with cell permeability in a concentration dependent manner.

Mechanistic insight into the reaction catalysed by bacterial type II dehydroquinases

DHQ2 (type II dehydroquinase), which is an essential enzyme in Helicobacter pylori and Mycobacterium tuberculosis and does not have any counterpart in humans, is recognized to be an attractive target for the development of new antibacterial agents. Computational and biochemical studies that help understand in atomic detail the catalytic mechanism of these bacterial enzymes are reported in the present paper. A previously unknown key role of certain conserved residues of these enzymes, as well as the structural changes responsible for triggering the release of the product from the active site, were identified. Asp89*/Asp88* from a neighbouring enzyme subunit proved to be the residue responsible for the deprotonation of the essential tyrosine to afford the catalytic tyrosinate, which triggers the enzymatic process. The essentiality of this residue is supported by results from site-directed mutagenesis. For H. pylori DHQ2, this reaction takes place through the assistance of a water molecule, whereas for M. tuberculosis DHQ2, the tyrosine is directly deprotonated by the aspartate residue. The participation of a water molecule in this deprotonation reaction is supported by solvent isotope effects and proton inventory studies. MD simulation studies provide details of the required motions for the catalytic turnover, which provides a complete overview of the catalytic cycle. The product is expelled from the active site by the essential arginine residue and after a large conformational change of a loop containing two conserved arginine residues (Arg109/Arg108 and Arg113/Arg112), which reveals a previously unknown key role for these residues. The present study highlights the key role of the aspartate residue whose blockage could be useful in the rational design of inhibitors and the mechanistic differences between both enzymes.

New insights into the catalytic mechanism of histidine phosphatases revealed by a functionally essential arginine residue within the active site of the Sts phosphatases

Sts (suppressor of T-cell receptor signalling)-1 and Sts-2 are HPs (histidine phosphatases) that negatively regulate TCR (T-cell receptor) signalling pathways, including those involved in cytokine production. HPs play key roles in such varied biological processes as metabolism, development and intracellular signalling. They differ considerably in their primary sequence and substrate specificity, but possess a catalytic core formed by an invariant quartet of active-site residues. Two histidine and two arginine residues cluster together within the HP active site and are thought to participate in a two-step dephosphorylation reaction. To date there has been little insight into any additional residues that might play an important functional role. In the present study, we identify and characterize an additional residue within the Sts phosphatases (Sts-1 Arg383 or Sts-2 Arg369) that is critical for catalytic activity and intracellular function. Mutation of Sts-1 Arg383 to an alanine residue compromises the enzyme's activity and renders Sts-1 unable to suppress TCR-induced cytokine induction. Of the multiple amino acids substituted for Arg383, only lysine partially rescues the catalytic activity of Sts-1. Although Sts-1 Arg383 is conserved in all Sts homologues, it is only conserved in one of the two sub-branches of HPs. The results of the present study highlight an essential role for Sts-1 phosphatase activity in regulating T-cell activation and add a new dimension of complexity to our understanding of HP catalytic activity.

Probing the origins of glutathione biosynthesis through biochemical analysis of glutamate-cysteine ligase and glutathione synthetase from a model photosynthetic prokaryote

Glutathione biosynthesis catalysed by GCL (glutamate-cysteine ligase) and GS (glutathione synthetase) is essential for maintaining redox homoeostasis and protection against oxidative damage in diverse eukaroytes and bacteria. This biosynthetic pathway probably evolved in cyanobacteria with the advent of oxygenic photosynthesis, but the biochemical characteristics of progenitor GCLs and GSs in these organisms are largely unexplored. In the present study we examined SynGCL and SynGS from Synechocystis sp. PCC 6803 using steady-state kinetics. Although SynGCL shares ~15% sequence identity with the enzyme from plants and α-proteobacteria, sequence comparison suggests that these enzymes share similar active site residues. Biochemically, SynGCL lacks the redox regulation associated with the plant enzymes and functions as a monomeric protein, indicating that evolution of redox regulation occurred later in the green lineage. Site-directed mutagenesis of SynGCL establishes this enzyme as part of the plant-like GCL family and identifies a catalytically essential arginine residue, which is structurally conserved across all forms of GCLs, including those from non-plant eukaryotes and γ-proteobacteria. A reaction mechanism for the synthesis of γ-glutamylcysteine by GCLs is proposed. Biochemical and kinetic analysis of SynGS reveals that this enzyme shares properties with other prokaryotic GSs. Initial velocity and product inhibition studies used to examine the kinetic mechanism of SynGS suggest that it and other prokaryotic GSs uses a random ter-reactant mechanism for the synthesis of glutathione. The present study provides new insight on the molecular mechanisms and evolution of glutathione biosynthesis; a key process required for enhancing bioenergy production in photosynthetic organisms.

Resolving the Negative Potential Side (n-side) Water-accessible Proton Pathway of F-type ATP Synthase by Molecular Dynamics Simulations

The rotation of F(1)F(o)-ATP synthase is powered by the proton motive force across the energy-transducing membrane. The protein complex functions like a turbine; the proton flow drives the rotation of the c-ring of the transmembrane F(o) domain, which is coupled to the ATP-producing F(1) domain. The hairpin-structured c-protomers transport the protons by reversible protonation/deprotonation of a conserved Asp/Glu at the outer transmembrane helix (TMH). An open question is the proton transfer pathway through the membrane at atomic resolution. The protons are thought to be transferred via two half-channels to and from the conserved cAsp/Glu in the middle of the membrane. By molecular dynamics simulations of c-ring structures in a lipid bilayer, we mapped a water channel as one of the half-channels. We also analyzed the suppressor mutant cP24D/E61G in which the functional carboxylate is shifted to the inner TMH of the c-protomers. Current models concentrating on the "locked" and "open" conformations of the conserved carboxylate side chain are unable to explain the molecular function of this mutant. Our molecular dynamics simulations revealed an extended water channel with additional water molecules bridging the distance of the outer to the inner TMH. We suggest that the geometry of the water channel is an important feature for the molecular function of the membrane part of F(1)F(o)-ATP synthase. The inclination of the proton pathway isolates the two half-channels and may contribute to a favorable clockwise rotation in ATP synthesis mode.

Phosphorylation of the Kinase Interaction Motif in Mitogen-activated Protein (MAP) Kinase Phosphatase-4 Mediates Cross-talk between Protein Kinase A and MAP Kinase Signaling Pathways

MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo.

Visualizing Adenylate Kinase Catalysis Through the X-ray Lens

∗ Conformational dynamics of Adenylate kinase is a key to understand the catalysis of this enzyme: a phosphoryl transfer reaction. Adenylate kinases maintain the level of nucleotides by transferring a phosphoryl group from ATP to AMP, which results in 2 ADPs in equilibrium. The active site locates where the ATP-lid and AMP-lid meet on top of the core domain. It has been proposed that various kinds of conformational stages exist by opening and closing the ATP- and the AMP-lids. However, only 4 characteristic types of structures so far have been reported: Open (without substrates, both lids open), Closed (with inhibitor, both lids closed), the AMP domain closed (with AMP, ATP-lid open without substrate) and the ATP domain closed (mutant Adkyst with ATP, AMP-lid open without substrate). We have determined crystal structures of AquifexAdenylate kinases with various substrates at high resolution up to 1.24 Å revealing conformational substates that include 1) a quasi-opened ATP-lid structure with substrates bound to both lids; 2) different conformations of substrates coinciding with movement of essential arginine residues; 3) a partially occupied metaphosphate intermediate structure. ∗ From these snapshots we suggest the role of arginine side chains in the active site with respect to the phosphoryl transfer step. By linking a series of these structures, we visualize the choreography of adenylate kinase catalysis in crystallographic view. NMR experiments characterizing the dynamics between these substates buttress our mechanistic interpretation of the x-ray snapshots.

Essential arginine residue of the Fo-a subunit in FoF1-ATP synthase has a role to prevent the proton shortcut without c-ring rotation in the Fo proton channel

In F(o)F(1) (F(o)F(1)-ATP synthase), proton translocation through F(o) drives rotation of the oligomer ring of F(o)-c subunits (c-ring) relative to F(o)-a. Previous reports have indicated that a conserved arginine residue in F(o)-a plays a critical role in the proton transfer at the F(o)-a/c-ring interface. Indeed, we show in the present study that thermophilic F(o)F(1s) with substitution of this arginine (aR169) to other residues cannot catalyse proton-coupled reactions. However, mutants with substitution of this arginine residue by a small (glycine, alanine, valine) or acidic (glutamate) residue mediate the passive proton translocation. This translocation requires an essential carboxy group of F(o)-c (cE56) since the second mutation (cE56Q) blocks the translocation. Rotation of the c-ring is not necessary because the same arginine mutants of the 'rotation-impossible' (c(10)-a)F(o)F(1), in which the c-ring and F(o)-a are fused to a single polypeptide, also exhibits the passive proton translocation. The mutant (aR169G/Q217R), in which the arginine residue is transferred to putatively the same topological position in the F(o)-a structure, can block the passive proton translocation. Thus the conserved arginine residue in F(o)-a ensures proton-coupled c-ring rotation by preventing a futile proton shortcut.

Specific Interaction between Tomato HsfA1 and HsfA2 Creates Hetero-oligomeric Superactivator Complexes for Synergistic Activation of Heat Stress Gene Expression

In plants, a family of more than 20 heat stress transcription factors (Hsf) controls the expression of heat stress (hs) genes. There is increasing evidence for the functional diversification between individual members of the Hsf family fulfilling distinct roles in response to various environmental stress conditions and developmental signals. In response to hs, accumulation of both heat stress proteins (Hsp) and Hsfs is induced. In tomato, the physical interaction between the constitutively expressed HsfA1 and the hs-inducible HsfA2 results in synergistic transcriptional activation (superactivation) of hs gene expression. Here, we show that the interaction is strikingly specific and not observed with other class A Hsfs. Hetero-oligomerization of the two-component Hsfs is preferred to homo-oligomerization, and each Hsf in the HsfA1/HsfA2 hetero-oligomeric complex has its characteristic contribution to its function as superactivator. Distinct regions of the oligomerization domain are responsible for specific homo- and hetero-oligomeric interactions leading to the formation of hexameric complexes. The results are summarized in a model of assembly and function of HsfA1/A2 superactivator complexes in hs gene regulation.

Fibronectin Unfolding Revisited: Modeling Cell Traction-Mediated Unfolding of the Tenth Type-III Repeat

Fibronectin polymerization is essential for the development and repair of the extracellular matrix. Consequently, deciphering the mechanism of fibronectin fibril formation is of immense interest. Fibronectin fibrillogenesis is driven by cell-traction forces that mechanically unfold particular modules within fibronectin. Previously, mechanical unfolding of fibronectin has been modeled by applying tensile forces at the N- and C-termini of fibronectin domains; however, physiological loading is likely focused on the solvent-exposed RGD loop in the 10th type-III repeat of fibronectin (10FNIII), which mediates binding to cell-surface integrin receptors. In this work we used steered molecular dynamics to study the mechanical unfolding of 10FNIII under tensile force applied at this RGD site. We demonstrate that mechanically unfolding 10FNIII by pulling at the RGD site requires less work than unfolding by pulling at the N- and C- termini. Moreover, pulling at the N- and C-termini leads to 10FNIII unfolding along several pathways while pulling on the RGD site leads to a single exclusive unfolding pathway that includes a partially unfolded intermediate with exposed hydrophobic N-terminal β-strands – residues that may facilitate fibronectin self-association. Additional mechanical unfolding triggers an essential arginine residue, which is required for high affinity binding to integrins, to move to a position far from the integrin binding site. This cell traction-induced conformational change may promote cell detachment after important partially unfolded kinetic intermediates are formed. These data suggest a novel mechanism that explains how cell-mediated forces promote fibronectin fibrillogenesis and how cell surface integrins detach from newly forming fibrils. This process enables cells to bind and unfold additional fibronectin modules – a method that propagates matrix assembly.