[Show abstract][Hide abstract] ABSTRACT: Streptococcus sanguinis is a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system.
An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets. S.
sanguinis expresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased
virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA
(SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 Å and 2.0 Å resolution, respectively. We identified
a conserved sequence Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers.
Further, we propose that sequence insertions in closely related family members contribute to the modulation of structural
and functional properties, including the quaternary structure, the tertiary structure, and the ligand binding site.
Preview · Article · Feb 2016 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone
oxidoreductase, SQR). Aerobically the bacterium synthesizes SQR as part of its respiratory chain whereas under microaerophilic
conditions the quinol:fumarate reductase (QFR) can be utilized. All complex II enzymes harbor a covalently bound FAD cofactor
that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin
linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the
covalent flavin bond or how it binds the flavoprotein subunit of complex II remains unknown. Using photocrosslinking we report
the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds
to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain
orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site which facilitates
formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs
participate in complex II maturation.
Preview · Article · Dec 2015 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Orthosomycins are oligosaccharide antibiotics that include avilamycin, everninomicin, and hygromycin B and are hallmarked by a rigidifying interglycosidic spirocyclic ortho-δ-lactone (orthoester) linkage between at least one pair of carbohydrates. A subset of orthosomycins additionally contain a carbohydrate capped by a methylenedioxy bridge. The orthoester linkage is necessary for antibiotic activity but rarely observed in natural products. Orthoester linkage and methylenedioxy bridge biosynthesis require similar oxidative cyclizations adjacent to a sugar ring. We have identified a conserved group of nonheme iron, α-ketoglutarate-dependent oxygenases likely responsible for this chemistry. High-resolution crystal structures of the EvdO1 and EvdO2 oxygenases of everninomicin biosynthesis, the AviO1 oxygenase of avilamycin biosynthesis, and HygX of hygromycin B biosynthesis show how these enzymes accommodate large substrates, a challenge that requires a variation in metal coordination in HygX. Excitingly, the ternary complex of HygX with cosubstrate α-ketoglutarate and putative product hygromycin B identified an orientation of one glycosidic linkage of hygromycin B consistent with metal-catalyzed hydrogen atom abstraction from substrate. These structural results are complemented by gene disruption of the oxygenases evdO1 and evdMO1 from the everninomicin biosynthetic cluster, which demonstrate that functional oxygenase activity is critical for antibiotic production. Our data therefore support a role for these enzymes in the production of key features of the orthosomycin antibiotics.
No preview · Article · Aug 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: G-protein-coupled receptors (GPCRs) are essential mediators of information transfer in eukaryotic cells. Interactions between GPCRs and their binding partners modulate the signaling process. For example, the interaction between GPCR and cognate G protein initiates the signal, while the interaction with cognate arrestin terminates G-protein-mediated signaling. In visual signal transduction, arrestin-1 selectively binds to the phosphorylated light-activated GPCR rhodopsin to terminate rhodopsin signaling. Under physiological conditions, the rhodopsin-arrestin-1 interaction occurs in highly specialized disk membrane in which rhodopsin resides. This membrane is replaced with mimetics when working with purified proteins. While detergents are commonly used as membrane mimetics, most detergents denature arrestin-1, preventing biochemical studies of this interaction. In contrast, bicelles provide a suitable alternative medium. An advantage of bicelles is that they contain lipids, which have been shown to be necessary for normal rhodopsin-arrestin-1 interaction. Here we describe how to reconstitute rhodopsin into bicelles, and how bicelle properties affect the rhodopsin-arrestin-1 interaction.
Full-text · Article · Feb 2015 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Rhodopsin is a prototypical member of the G protein-coupled receptors (GPCRs). This photoreceptor is responsible for initiating the visual signaling transduction cascade upon interaction with its heterotrimeric G protein, transducin (Gt), after light activation. Like all transmembrane proteins, rhodopsin is embedded within a phospholipid bilayer. Many studies have proposed that the membrane composition of this bilayer is an important factor for receptor function during the activation process. Here we describe the methods and assays used to evaluate the function of purified and reconstituted rhodopsin in bicelles.
No preview · Article · Feb 2015 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: G protein activation by G protein coupled receptors (GPCRs) is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the alpha 5 (α5) helix in the G protein alpha subunit plays a major role during this activation process. However, the precise signaling pathway between the α5 helix and the GDP binding pocket remains elusive. Here, using structural, biochemical and computational techniques, we probed different residues around the α5 helix for their role in signaling. Our data showed that perturbing the F336 (α5) residue disturbs hydrophobic interactions with the β2-β3 strands and α1 helix, leading to high basal nucleotide exchange. However, mutations in β strands β5 and β6 do not perturb G protein activation. We have highlighted critical residues that leverage F336 as a relay. Conformational changes are transmitted starting from F336 via β2-β3/α1 to Switch I and the P-loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the α1 and α5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal α5 helix, mutation of F336 still leads to high basal exchange. This suggests that unlike receptor mediated activation, helix 5 rotation and translocation is not necessary for GDP release from the α subunit. Rather, destabilization of the backdoor region of the Gα subunit is sufficient for triggering the activation process.
[Show abstract][Hide abstract] ABSTRACT: Concatenation of engineered biocatalysts into multistep pathways markedly increases their utility, but the development of generalizable assembly methods remains a major challenge. Herein we evaluate 'bioretrosynthesis', which is an application of the retrograde evolution hypothesis, for biosynthetic pathway construction. To test bioretrosynthesis, we engineered a pathway for synthesis of the antiretroviral nucleoside analog didanosine (2',3'-dideoxyinosine). Applying both directed evolution- and structure-based approaches, we began pathway construction with a retro-extension from an engineered purine nucleoside phosphorylase and evolved 1,5-phosphopentomutase to accept the substrate 2,3-dideoxyribose 5-phosphate with a 700-fold change in substrate selectivity and threefold increased turnover in cell lysate. A subsequent retrograde pathway extension, via ribokinase engineering, resulted in a didanosine pathway with a 9,500-fold change in nucleoside production selectivity and 50-fold increase in didanosine production. Unexpectedly, the result of this bioretrosynthetic step was not a retro-extension from phosphopentomutase but rather the discovery of a fortuitous pathway-shortening bypass via the engineered ribokinase.
Full-text · Article · Mar 2014 · Nature Chemical Biology
[Show abstract][Hide abstract] ABSTRACT: Receptor-mediated activation of the Gα subunit of heterotrimeric G proteins requires allosteric communication between the receptor-binding and the guanine nucleotide-binding sites, which are separated by over 30 Å. Structural changes in the allosteric network connecting these sites are predicted to be transient in the wild-type Gα subunit, making studies of these connections challenging. In the current work, site-directed mutants that alter the energy barriers between the activation states are used as tools to better understand transient features of allosteric signaling in the Gα subunit. The observed differences in relative receptor affinity for intact Gαi1 subunits versus C-terminal Gαi1 peptides harboring the K345L mutation are consistent with this mutation modulating the allosteric network in the protein subunit. Measurement of nucleotide exchange rates, affinity for meta II, and thermostability suggest that the K345L Gαi1 variant has reduced stability in both the GDP-bound and nucleotide-free states as compared to wild-type, but exhibits similar stability in the GTPγS-bound state. High-resolution X-ray crystal structures reveal conformational changes accompanying the destabilization of the GDP-bound state. Of these, a new conformation for Switch I was stabilized by an ionic interaction with the P-loop. Further site-directed mutagenesis suggests that this interaction between Switch I and the P-loop is important for receptor-mediated nucleotide exchange in the wild-type Gαi subunit.
Full-text · Article · Mar 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The serine-rich repeat glycoproteins of Gram-positive bacteria comprise a large family of cell wall proteins. Streptococcus agalactiae (group B streptococcus, GBS) expresses either Srr1 or Srr2 on its surface, depending on the strain. Srr1 has recently been shown to bind fibrinogen, and this interaction contributes to the pathogenesis of GBS meningitis. Although strains expressing Srr2 appear to be hypervirulent, no ligand for this adhesin has been described. We now demonstrate that Srr2 also binds human fibrinogen and that this interaction promotes GBS attachment to endothelial cells. Recombinant Srr1 and Srr2 bound fibrinogen in vitro, with affinities of KD = 2.1 × 10−5 and 3.7 × 10−6
m, respectively, as measured by surface plasmon resonance spectroscopy. The binding site for Srr1 and Srr2 was localized to tandem repeats 6–8 of the fibrinogen Aα chain. The structures of both the Srr1 and Srr2 binding regions were determined and, in combination with mutagenesis studies, suggest that both Srr1 and Srr2 interact with a segment of these repeats via a “dock, lock, and latch” mechanism. Moreover, properties of the latch region may account for the increased affinity between Srr2 and fibrinogen. Together, these studies identify how greater affinity of Srr2 for fibrinogen may contribute to the increased virulence associated with Srr2-expressing strains.
No preview · Article · Oct 2013 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Respiratory processes often use quinone oxidoreduction to generate a transmembrane proton gradient, making the 2H+/2e- quinone chemistry important for ATP synthesis. There are a variety of quinones used as electron carriers between bioenergetic proteins, and some respiratory proteins can functionally interact with more than one quinone type. In the case of complex II homologs, which couple quinone chemistry to the interconversion of succinate and fumarate, the redox potentials of the biologically available ubiquinone and menaquinone aid in driving the chemical reaction in one direction. In the complex II homolog quinol:fumarate reductase, it has been demonstrated that menaquinol oxidation requires at least one proton shuttle, but many of the remaining mechanistic details of menaquinol oxidation are not fully understood and little is known about ubiquinone reduction. In the current study structural and computational studies suggest that the sequential removal of the two menaquinol protons may be accompanied by a rotation of the naphthoquinone ring to optimize the interaction with a second proton shuttling pathway. However, kinetic measurements of site-specific mutations of quinol:fumarate reductase variants show that ubiquinone reduction does not use the same pathway. Computational docking of ubiquinone followed by mutagenesis instead suggested redundant proton shuttles lining the ubiquinone binding site or from direct transfer from solvent. These data show that the quinone binding site provides an environment that allows multiple amino acid residues to participate in quinone oxidoreduction. This suggests that the quinone-binding site in complex II is inherently plastic and can robustly interact with different types of quinones.
No preview · Article · Jul 2013 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Solution NMR spectroscopy of labeled arrestin-1 was used to explore its interactions with dark-state phosphorylated rhodopsin (P-Rh), phosphorylated opsin (P-opsin), unphosphorylated light-activated rhodopsin (Rh*), and phosphorylated light-activated rhodopsin (P-Rh*). Distinct sets of arrestin-1 elements were seen to be engaged by Rh* and inactive P-Rh, which induced conformational changes that differed from those triggered by binding of P-Rh*. Although arrestin-1 affinity for Rh* was seen to be low (K(D) > 150 μM), its affinity for P-Rh (K(D) ∼80 μM) was comparable to the concentration of active monomeric arrestin-1 in the outer segment, suggesting that P-Rh generated by high-gain phosphorylation is occupied by arrestin-1 under physiological conditions and will not signal upon photo-activation. Arrestin-1 was seen to bind P-Rh* and P-opsin with fairly high affinity (K(D) of ∼50 and 800 nM, respectively), implying that arrestin-1 dissociation is triggered only upon P-opsin regeneration with 11-cis-retinal, precluding noise generated by opsin activity. Based on their observed affinity for arrestin-1, P-opsin and inactive P-Rh very likely affect the physiological monomer-dimer-tetramer equilibrium of arrestin-1, and should therefore be taken into account when modeling photoreceptor function. The data also suggested that complex formation with either P-Rh* or P-opsin results in a global transition in the conformation of arrestin-1, possibly to a dynamic molten globule-like structure. We hypothesize that this transition contributes to the mechanism that triggers preferential interactions of several signaling proteins with receptor-activated arrestins.
Full-text · Article · Dec 2012 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Arrestin-1 preferentially binds active phosphorylated rhodopsin. Previously, a mutant with enhanced binding to unphosphorylated active rhodopsin (Rh*) was shown to partially compensate for lack of rhodopsin phosphorylation in vivo. Here we show that reengineering of the receptor-binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability. In mammals, arrestin-1 readily self-associates at physiological concentrations. The biological role of this phenomenon can only be elucidated by replacing wild type arrestin-1 in living animals with a non-oligomerizing mutant retaining all other functions. We demonstrate that constitutively monomeric forms of arrestin-1 are sufficiently stable for in vivo expression. We also tested the idea that individual functions of arrestin-1 can be independently manipulated to generate mutants with the desired combinations of functional characteristics. We show that this approach is feasible: stable forms of arrestin-1 with high Rh* binding can be generated with or without the ability to self-associate. These novel molecular tools open the possibility of testing of the biological role of arrestin-1 self-association, and pave the way to elucidation of full potential of compensational approach to gene therapy of gain-of-function receptor mutations.
Full-text · Article · Dec 2012 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Acetate kinases (ACKs) are members of the acetate and sugar kinase/hsp70/actin (ASKHA) superfamily and catalyze the reversible phosphorylation of acetate, with ADP/ATP the most common phosphoryl acceptor/donor. While prokaryotic ACKs have been the subject of extensive biochemical and structural characterization, there is a comparative paucity of information on eukaryotic ACKs, and prior to this report, no structure of an ACK of eukaryotic origin was available. We determined the structures of ACKs from the eukaryotic pathogens Entamoeba histolytica and Cryptococcus neoformans. Each active site is located at an interdomain interface, and the acetate and phosphate binding pockets display sequence and structural conservation with their prokaryotic counterparts. Interestingly, the E. histolytica ACK has previously been shown to be pyrophosphate (PP(i))-dependent, and is the first ACK demonstrated to have this property. Examination of its structure demonstrates how subtle amino acid substitutions within the active site have converted cosubstrate specificity from ATP to PP(i) while retaining a similar backbone conformation. Differences in the angle between domains surrounding the active site suggest that interdomain movement may accompany catalysis. Taken together, these structures are consistent with the eukaryotic ACKs following a similar reaction mechanism as is proposed for the prokaryotic homologs.
Full-text · Article · Nov 2012 · Journal of Structural Biology
[Show abstract][Hide abstract] ABSTRACT: Over a decade has passed since the elucidation of the first X-ray crystal structure of any complex II homolog. In the intervening time, the structures of five additional integral-membrane complex II enzymes and three homologs of the soluble domain have been determined. These structures have provided a framework for the analysis of enzymological studies of complex II superfamily enzymes, and have contributed to detailed proposals for reaction mechanisms at each of the two enzyme active sites, which catalyze dicarboxylate and quinone oxidoreduction, respectively. This review focuses on how structural data have augmented our understanding of catalysis by the superfamily. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.
[Show abstract][Hide abstract] ABSTRACT: Complex II couples oxidoreduction of succinate and fumarate at one active site with that of quinol/quinone at a second distinct
active site over 40 Å away. This process links the Krebs cycle to oxidative phosphorylation and ATP synthesis. The pathogenic
mutation or inhibition of human complex II or its assembly factors is often associated with neurodegeneration or tumor formation
in tissues derived from the neural crest. This brief overview of complex II correlates the clinical presentations of a large
number of symptom-associated alterations in human complex II activity and assembly with the biochemical manifestations of
similar alterations in the complex II homologs from Escherichia coli. These analyses provide clues to the molecular basis for diseases associated with aberrant complex II function.
Preview · Article · Aug 2012 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Prokaryotic phosphopentomutases (PPMs) are di-Mn(2+) enzymes that catalyze the interconversion of α-D-ribose 5-phosphate and α-D-ribose 1-phosphate at an active site located between two independently folded domains. These prokaryotic PPMs belong to the alkaline phosphatase superfamily, but previous studies of Bacillus cereus PPM suggested adaptations of the conserved alkaline phosphatase catalytic cycle. Notably, B. cereus PPM engages substrates when the active site nucleophile, Thr-85, is phosphorylated. Further, the phosphoenzyme is stable throughout purification and crystallization. In contrast, alkaline phosphatase engages substrates when the active site nucleophile is dephosphorylated, and the phosphoenzyme reaction intermediate is only stably trapped in a catalytically compromised enzyme. Studies were undertaken to understand the divergence of these mechanisms. Crystallographic and biochemical investigations of the PPM(T85E) phosphomimetic variant and the neutral corollary PPM(T85Q) determined that the side chain of Lys-240 underwent a change in conformation in response to active site charge, which modestly influenced the affinity for the small molecule activator α-D-glucose 1,6-bisphosphate. More strikingly, the structure of unphosphorylated B. cereus PPM revealed a dramatic change in the interdomain angle and a new hydrogen bonding interaction between the side chain of Asp-156 and the active site nucleophile, Thr-85. This hydrogen bonding interaction is predicted to align and activate Thr-85 for nucleophilic addition to α-D-glucose 1,6-bisphosphate, favoring the observed equilibrium phosphorylated state. Indeed, phosphorylation of Thr-85 is severely impaired in the PPM(D156A) variant even under stringent activation conditions. These results permit a proposal for activation of PPM and explain some of the essential features that distinguish between the catalytic cycles of PPM and alkaline phosphatase.