Search tips
Search criteria

Results 1-25 (4793)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
more »
6.  Mutational Analysis of the C8-Guanine Adduct of the Environmental Carcinogen 3-Nitrobenzanthrone in Human Cells: Critical Roles of DNA Polymerases η and κ and Rev1 in Error-Prone Translesion Synthesis 
Biochemistry  2014;53(32):5323-5331.
3-Nitrobenzanthrone (3-NBA), a potent mutagen and suspected human carcinogen, is a common environmental pollutant. The genotoxicity of 3-NBA has been associated with its ability to form DNA adducts, including N-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). To investigate the molecular mechanism of C8-dG-ABA mutagenesis in human cells, we have replicated a plasmid containing a single C8-dG-ABA in human embryonic kidney 293T (HEK293T) cells, which yielded 14% mutant progeny. The major types of mutations induced by C8-dG-ABA were G → T > G → A > G → C. siRNA knockdown of the translesion synthesis (TLS) DNA polymerases (pols) in HEK293T cells indicated that pol η, pol κ, pol ι, pol ζ, and Rev1 each have a role in replication across this adduct. The extent of TLS was reduced with each pol knockdown, but the largest decrease (of ∼55% reduction) in the level of TLS occurred in cells with knockdown of pol ζ. Pol η and pol κ were considered the major contributors of the mutagenic TLS, because the mutation frequency (MF) decreased by 70%, when these pols were simultaneously knocked down. Rev1 also is important for mutagenesis, as reflected by the 60% reduction in MF upon Rev1 knockdown, but it probably plays a noncatalytic role by physically interacting with the other two Y-family pols. In contrast, pol ζ appeared to be involved in the error-free bypass of the lesion, because MF increased by 60% in pol ζ knockdown cells. These results provide important mechanistic insight into the bypass of the C8-dG-ABA adduct.
PMCID: PMC4139159  PMID: 25080294
7.  Molecular Features of Product Release for the PKA Catalytic Cycle 
Biochemistry  2014;54(1):2-10.
Although ADP release is the rate limiting step in product turnover by protein kinase A, the steps and motions involved in this process are not well resolved. Here we report the apo and ADP bound structures of the myristylated catalytic subunit of PKA at 2.9 and 3.5 Å resolution, respectively. The ADP bound structure adopts a conformation that does not conform to the previously characterized open, closed, or intermediate states. In the ADP bound structure, the C-terminal tail and Gly-rich loop are more closed than in the open state adopted in the apo structure but are also much more open than the intermediate or closed conformations. Furthermore, ADP binds at the active site with only one magnesium ion, termed Mg2 from previous structures. These structures thus support a model where ADP release proceeds through release of the substrate and Mg1 followed by lifting of the Gly-rich loop and disengagement of the C-terminal tail. Coupling of these two structural elements with the release of the first metal ion fills in a key step in the catalytic cycle that has been missing and supports an ensemble of correlated conformational states that mediate the full catalytic cycle for a protein kinase.
PMCID: PMC4295794  PMID: 25077557
8.  Cathepsin L Mediates the Degradation of Novel APP C-Terminal Fragments 
Biochemistry  2015;54(18):2806-2816.
Alzheimer's disease (AD) is characterized by the deposition of amyloid β (Aβ), a peptide generated from proteolytic processing of its precursor, amyloid precursor protein (APP). Canonical APP proteolysis occurs via α-, β-, and γ-secretases. APP is also actively degraded by protein degradation systems. By pharmacologically inhibiting protein degradation with ALLN, we observed an accumulation of several novel APP C-terminal fragments (CTFs). The two major novel CTFs migrated around 15 and 25 kDa and can be observed across multiple cell types. The process was independent of cytotoxicity or protein synthesis. We further determine that the accumulation of the novel CTFs is not mediated by proteasome or calpain inhibition, but by cathepsin L inhibition. Moreover, these novel CTFs are not generated by an increased amount of BACE. Here, we name the CTF of 25 kDa as η-CTF (eta-CTF). Our data suggest that under physiological conditions, a subset of APP undergoes alternative processing and the intermediate products, the 15 kDa CTFs, and the η-CTFs aret rapidly degraded and/or processed via the protein degradation machinery, specifically, cathepsin L.
PMCID: PMC4521409  PMID: 25910068
9.  Tunable Control of Polyproline Helix (PPII) Structure via Aromatic Electronic Effects: An Electronic Switch of Polyproline Helix 
Biochemistry  2014;53(32):5307-5314.
Aromatic rings exhibit defined interactions via the unique aromatic π face. Aromatic amino acids interact favorably with proline residues via both the hydrophobic effect and aromatic–proline interactions, C−H/π interactions between the aromatic π face and proline ring C–H bonds. The canonical aromatic amino acids Trp, Tyr, and Phe strongly disfavor a polyproline helix (PPII) when they are present in proline-rich sequences because of the large populations of cis amide bonds induced by favorable aromatic–proline interactions (aromatic–cis-proline and proline–cis-proline–aromatic interactions). We demonstrate the ability to tune polyproline helix conformation and cis–trans isomerism in proline-rich sequences using aromatic electronic effects. Electron-rich aromatic residues strongly disfavor polyproline helix and exhibit large populations of cis amide bonds, while electron-poor aromatic residues exhibit small populations of cis amide bonds and favor polyproline helix. 4-Aminophenylalanine is a pH-dependent electronic switch of polyproline helix, with cis amide bonds favored as the electron-donating amine, but trans amide bonds and polyproline helix preferred as the electron-withdrawing ammonium. Peptides with block proline–aromatic PPXPPXPPXPP sequences exhibited electronically switchable pH-dependent structures. Electron-poor aromatic amino acids provide special capabilities to integrate aromatic residues into polyproline helices and to serve as the basis of aromatic electronic switches to change structure.
PMCID: PMC4139158  PMID: 25075447
10.  Requirements for Mutant and Wild-Type Prion Protein Misfolding In Vitro 
Biochemistry  2015;54(5):1180-1187.
Misfolding of the prion protein (PrP) plays a central role in the pathogenesis of infectious, sporadic, and inherited prion diseases. Here we use a chemically defined prion propagation system to study misfolding of the pathogenic PrP mutant D177N in vitro. This mutation causes PrP to misfold spontaneously in the absence of cofactor molecules in a process dependent on time, temperature, pH, and intermittent sonication. Spontaneously misfolded mutant PrP is able to template its unique conformation onto wild-type PrP substrate in a process that requires a phospholipid activity distinct from that required for the propagation of infectious prions. Similar results were obtained with a second pathogenic PrP mutant, E199K, but not with the polymorphic substitution M128V. Moreover, wild-type PrP inhibits mutant PrP misfolding in a dose-dependent manner, and cofactor molecules can antagonize this effect. These studies suggest that interactions between mutant PrP, wild-type PrP, and other cellular factors may control the rate of PrP misfolding in inherited prion diseases.
PMCID: PMC4520438  PMID: 25584902
11.  Properties of the Mechanosensitive Channel MscS Pore Revealed by Tryptophan Scanning Mutagenesis 
Biochemistry  2015;54(29):4519-4530.
Bacterial mechanosensitive channels gate when the transmembrane turgor rises to levels that compromise the structural integrity of the cell wall. Gating creates a transient large diameter pore that allows hydrated solutes to pass from the cytoplasm at rates close to those of diffusion. In the closed conformation, the channel limits transmembrane solute movement, even that of protons. In the MscS crystal structure (Protein Data Bank entry 2oau), a narrow, hydrophobic opening is visible in the crystal structure, and it has been proposed that a vapor lock created by the hydrophobic seals, L105 and L109, is the barrier to water and ions. Tryptophan scanning mutagenesis has proven to be a highly valuable tool for the analysis of channel structure. Here Trp residues were introduced along the pore-forming TM3a helix and in selected other parts of the protein. Mutants were investigated for their expression, stability, and activity and as fluorescent probes of the physical properties along the length of the pore. Most Trp mutants were expressed at levels similar to that of the parent (MscS YFF) and were stable as heptamers in detergent in the presence and absence of urea. Fluorescence data suggest a long hydrophobic region with low accessibility to aqueous solvents, extending from L105/L109 to G90. Steady-state fluorescence anisotropy data are consistent with significant homo-Förster resonance energy transfer between tryptophan residues from different subunits within the narrow pore. The data provide new insights into MscS structure and gating.
PMCID: PMC4519979  PMID: 26126964
12.  Analytical Comparison of Natural and Pharmaceutical Ventricular Myosin Activators 
Biochemistry  2014;53(32):5298-5306.
Ventricular myosin (βMys) is the motor protein in cardiac muscle generating force using ATP hydrolysis free energy to translate actin. In the cardiac muscle sarcomere, myosin and actin filaments interact cyclically and undergo rapid relative translation facilitated by the low duty cycle motor. It contrasts with high duty cycle processive myosins for which persistent actin association is the priority. The only pharmaceutical βMys activator, omecamtive mecarbil (OM), upregulates cardiac contractility in vivo and is undergoing testing for heart failure therapy. In vitro βMys step-size, motility velocity, and actin-activated myosin ATPase were measured to determine duty cycle in the absence and presence of OM. A new parameter, the relative step-frequency, was introduced and measured to characterize βMys motility due to the involvement of its three unitary step-sizes. Step-size and relative step-frequency were measured using the Qdot assay. OM decreases motility velocity 10-fold without affecting step-size, indicating a large increase in duty cycle converting βMys to a near processive myosin. The OM conversion dramatically increases force and modestly increases power over the native βMys. Contrasting motility modification due to OM with that from the natural myosin activator, specific βMys phosphorylation, provides insight into their respective activation mechanisms and indicates the boilerplate screening characteristics desired for pharmaceutical βMys activators. New analytics introduced here for the fast and efficient Qdot motility assay create a promising method for high-throughput screening of motor proteins and their modulators.
PMCID: PMC4139156  PMID: 25068717
13.  NMR Solution Structure of the Terminal Immunoglobulin-like Domain from the Leptospira Host-Interacting Outer Membrane Protein, LigB 
Biochemistry  2014;53(32):5249-5260.
A number of surface proteins specific to pathogenic strains of Leptospira have been identified. The Lig protein family has shown promise as a marker in typing leptospiral isolates for pathogenesis and as an antigen in vaccines. We used NMR spectroscopy to solve the solution structure of the twelfth immunoglobulin-like (Ig-like) repeat domain from LigB (LigB-12). The fold is similar to that of other bacterial Ig-like domains and comprised mainly of β-strands that form a β-sandwich based on a Greek-key folding arrangement. Based on sequence analysis and conservation of structurally important residues, homology models for the other LigB Ig-like domains were generated. The set of LigB models illustrates the electrostatic differences between the domains as well as the possible interactions between neighboring domains. Understanding the structure of the extracellular portion of LigB and related proteins is important for developing diagnostic methods and new therapeutics directed toward leptospirosis.
PMCID: PMC4139157  PMID: 25068811
14.  Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase from Thermotoga maritima: Carbon Transfer without Covalent Pyrimidine Activation 
Biochemistry  2014;53(32):5199-5207.
Thymidylate, a vital DNA precursor, is synthesized by thymidylate synthases (TSs). A second class of TSs, encoded by the thyX gene, is found in bacteria and a few other microbes and is especially widespread in anaerobes. TS encoded by thyX requires a flavin adenine dinucleotide prosthetic group for activity. In the oxidative half-reaction, the reduced flavin is oxidized by 2′-deoxyuridine 5′-monophosphate (dUMP) and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF), synthesizing 2′-deoxythymidine 5′-monophosphate (dTMP). dTMP synthesis is a complex process, requiring the enzyme to promote carbon transfer, probably by increasing the nucleophilicity of dUMP and the electrophilicity of CH2THF, and reduction of the transferred carbon. The mechanism of the oxidative half-reaction was investigated by transient kinetics. Two intermediates were detected, the first by a change in the flavin absorbance spectrum in stopped-flow experiments and the second by the transient disappearance of deoxynucleotide in acid quenching experiments. The effects of substrate analogues and the behavior of mutated enzymes on these reactions lead to the conclusion that activation of dUMP does not occur through a Michael-like addition, the mechanism for the activation analogous with that of the flavin-independent TS. Rather, we propose that the nucleophilicity of dUMP is enhanced by electrostatic polarization upon binding to the active site. This conclusion rationalizes many of our observations, for instance, the markedly slower reactions when two arginine residues that hydrogen bond with the uracil moiety of dUMP were mutated to alanine. The activation of dUMP by polarization is consistent with the majority of the published data on ThyX and provides a testable mechanistic hypothesis.
PMCID: PMC4139161  PMID: 25068636
15.  X-ray Crystal Structure of Bovine 3 Glu-Osteocalcin 
Biochemistry  2013;52(47):8387-8392.
The 3 Glu form of osteocalcin (3 Glu-OCN) is increased in serum during low vitamin K intake or oral anticoagulant use (warfarin). Previous reports using circular dichroism show it is less structured than 3 Gla Ca2+-osteocalcin and does not bind strongly to bone mineral. Recent studies have suggested a role for 3 Glu-OCN as a potential regulator of glucose metabolism. A G-protein-coupled receptor, GPRC6a, found in the pancreas and testes was identified as the putative osteocalcin receptor. The purpose of this study is to determine the high-resolution structure of bovine 3 Glu-OCN, using X-ray crystallography, to understand molecular interactions with mineral and the GPRC6a receptor. Diffraction quality crystals of thermally decarboxylated bovine osteocalcin were grown, and the crystal structure was determined to 1.88 Å resolution. The final refined structure contained residues 17–47 and, like 3 Gla Ca2+-OCN, consisted of three α-helices surrounding a hydrophobic core, a C23–C29 disulfide bond between two of the helices, and no bound Ca2+. Thus, the helical structure of 3 Glu-OCN is Ca2+-independent but similar to that of 3 Gla Ca2+-OCN. A reduced level of mineral binding could result from a lower number of Ca2+ coordinating ligands on 3 Glu-OCN. The structure suggests the GPRC6a receptor may respond to helical osteocalcin and will aid in providing molecular mechanistic insight into the role of 3 Glu-OCN in glucose homeostasis.
PMCID: PMC4517604  PMID: 24138653
16.  The Three-Dimensional Structure of Bovine Calcium Ion-Bound Osteocalcin Using 1H NMR Spectroscopy† 
Biochemistry  2003;42(25):7769-7779.
Structural information on osteocalcin or other noncollagenous bone proteins is very limited. We have solved the three-dimensional structure of calcium bound osteocalcin using 1H 2D NMR techniques and proposed a mechanism for mineral binding. The protons in the 49 amino acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. Distance constraints, dihedral angle constraints, hydrogen bonds, and 1H and 13C chemical shifts were all used to calculate a family of 13 structures. The tertiary structure of the protein consisted of an unstructured N terminus and a C-terminal loop (residues 16–49) formed by long-range hydrophobic interactions. Elements of secondary structure within residues 16–49 include type III turns (residues 20–25) and two α-helical regions (residues 27–35 and 41–44). The three Gla residues project from the same face of the helical turns and are surface exposed. The genetic algorithm–molecular dynamics simulation approach was used to place three calcium atoms on the NMR-derived structure. One calcium atom was coordinated by three side chain oxygen atoms, two from Asp30, and one from Gla24. The second calcium atom was coordinated to four oxygen atoms, two from the side chain in Gla 24, and two from the side chain of Gla 21. The third calcium atom was coordinated to two oxygen atoms of the side chain of Gla17. The best correlation of the distances between the uncoordinated Gla oxygen atoms is with the intercalcium distance of 9.43 Å in hydroxyapatite. The structure may provide further insight into the function of osteocalcin.
PMCID: PMC4517610  PMID: 12820886
17.  Nox4: A Hydrogen Peroxide-Generating Oxygen Sensor 
Biochemistry  2014;53(31):5111-5120.
Nox4 is an oddity among members of the Nox family of NADPH oxidases [seven isoenzymes that generate reactive oxygen species (ROS) from molecular oxygen] in that it is constitutively active. All other Nox enzymes except for Nox4 require upstream activators, either calcium or organizer/activator subunits (p47phox, NOXO1/p67phox, and NOXA1). Nox4 may also be unusual as it reportedly releases hydrogen peroxide (H2O2) in contrast to Nox1–Nox3 and Nox5, which release superoxide, although this result is controversial in part because of possible membrane compartmentalization of superoxide, which may prevent detection. Our studies were undertaken (1) to identify the Nox4 ROS product using a membrane-free, partially purified preparation of Nox4 and (2) to test the hypothesis that Nox4 activity is acutely regulated not by activator proteins or calcium, but by cellular pO2, allowing it to function as an O2 sensor, the output of which is signaling H2O2. We find that approximately 90% of the electron flux through isolated Nox4 produces H2O2 and 10% forms superoxide. The kinetic mechanism of H2O2 formation is consistent with a mechanism involving binding of one oxygen molecule, which is then sequentially reduced by the heme in two one-electron reduction steps first to form a bound superoxide intermediate and then H2O2; kinetics are not consistent with a previously proposed internal superoxide dismutation mechanism involving two oxygen binding/reduction steps for each H2O2 formed. Critically, Nox4 has an unusually high Km for oxygen (∼18%), similar to the values of known oxygen-sensing enzymes, compared with a Km of 2–3% for Nox2, the phagocyte NADPH oxidase. This allows Nox4 to generate H2O2 as a function of oxygen concentration throughout a physiological range of pO2 values and to respond rapidly to changes in pO2.
PMCID: PMC4131900  PMID: 25062272
18.  A Novel CXCR4-Selective High-Affinity Fluorescent Probe and Its Application in Competitive Binding Assays 
Biochemistry  2014;53(30):4881-4883.
We recently developed a new, rapid, and specific bioassay system that employs a fluorescent probe fabricated from our discovered CXCR4-specific ligand DV1. This new probe sensitively and selectively blocks the binding of native and synthetic ligands to CXCR4 at nanomolar levels, with a capability comparable to that seen with a conventional CXCR4 antibody. This nonradioactive, direct, and CXCR4-specific high-affinity screening system provides a new platform for CXCR4-targeted drug screening, as well as for the development of new probes for other GPCRs.
PMCID: PMC4144712  PMID: 25058910
19.  Conserved Residues of the Human Mitochondrial Holocytochrome c Synthase Mediate Interactions with Heme 
Biochemistry  2014;53(32):5261-5271.
C-type cytochromes are distinguished by the covalent attachment of a heme cofactor, a modification that is typically required for its subsequent folding, stability, and function. Heme attachment takes place in the mitochondrial intermembrane space and, in most eukaryotes, is mediated by holocytochrome c synthase (HCCS). HCCS is the primary component of the eukaryotic cytochrome c biogenesis pathway, known as System III. The catalytic function of HCCS depends on its ability to coordinate interactions between its substrates: heme and cytochrome c. Recent advancements in the recombinant expression and purification of HCCS have facilitated comprehensive analyses of the roles of conserved residues in HCCS, as demonstrated in this study. Previously, we proposed a four-step model describing HCCS-mediated cytochrome c assembly, identifying a conserved histidine residue (His154) as an axial ligand to the heme iron. In this study, we performed a systematic mutational analysis of 17 conserved residues in HCCS, and we provide evidence that the enzyme contains two heme-binding domains. Our data indicate that heme contacts mediated by residues within these domains modulate the dynamics of heme binding and contribute to the stability of the HCCS–heme–cytochrome c steady state ternary complex. While some residues are essential for initial heme binding (step 1), others impact the subsequent release of the holocytochrome c product (step 4). Certain HCCS mutants that were defective in heme binding were corrected for function by exogenous aminolevulinic acid (ALA, the precursor to heme). This chemical “correction” supports the proposed role of heme binding for the corresponding residues.
PMCID: PMC4139152  PMID: 25054239
20.  pH-Induced Conformational Change of IscU at Low pH Correlates with Protonation/Deprotonation of Two Conserved Histidine Residues 
Biochemistry  2014;53(32):5290-5297.
IscU, the scaffold protein for the major iron–sulfur cluster biosynthesis pathway in microorganisms and mitochondria (ISC pathway), plays important roles in the formation of [2Fe–2S] and [4Fe–4S] clusters and their delivery to acceptor apo-proteins. Our laboratory has shown that IscU populates two distinct, functionally relevant conformational states, a more structured state (S) and a more dynamic state (D), that differ by cis/trans isomerizations about two peptidyl-prolyl peptide bonds [Kim, J. H., Tonelli, M., and Markley, J. L. (2012) Proc. Natl. Acad. Sci. U.S.A., 109, 454–459. Dai Z., Tonelli, M., and Markley, J. L. (2012) Biochemistry, 51, 9595–9602. Cai, K., Frederick, R. O., Kim, J. H., Reinen, N. M., Tonelli, M., and Markley, J. L. (2013) J. Biol. Chem., 288, 28755–28770]. Here, we report our findings on the pH dependence of the D ⇄ S equilibrium for Escherichia coli IscU in which the D-state is stabilized at low and high pH values. We show that the lower limb of the pH dependence curve results from differences in the pKa values of two conserved histidine residues (His10 and His105) in the two states. The net proton affinity of His10 is about 50 times higher and that of His105 is 13 times higher in the D-state than in the S-state. The origin of the high limb of the D ⇄ S pH dependence remains to be determined. These results show that changes in proton inventory need to be taken into account in the steps in iron–sulfur cluster assembly and transfer that involve transitions of IscU between its S- and D-states.
PMCID: PMC4139155  PMID: 25055301
21.  G Protein-Coupled Receptor Kinase 2 (GRK2) and 5 (GRK5) Exhibit Selective Phosphorylation of the Neurotensin Receptor in Vitro 
Biochemistry  2015;54(28):4320-4329.
G protein-coupled receptor kinases (GRKs) play an important role in the desensitization of G protein-mediated signaling of G protein-coupled receptors (GPCRs). The level of interest in mapping their phosphorylation sites has increased because recent studies suggest that the differential pattern of receptor phosphorylation has distinct biological consequences. In vitro phosphorylation experiments using well-controlled systems are useful for deciphering the complexity of these physiological reactions and understanding the targeted event. Here, we report on the phosphorylation of the class A GPCR neurotensin receptor 1 (NTSR1) by GRKs under defined experimental conditions afforded by nanodisc technology. Phosphorylation of NTSR1 by GRK2 was agonist-dependent, whereas phosphorylation by GRK5 occurred in an activation-independent manner. In addition, the negatively charged lipids in the immediate vicinity of NTSR1 directly affect phosphorylation by GRKs. Identification of phosphorylation sites in agonist-activated NTSR1 revealed that GRK2 and GRK5 target different residues located on the intracellular receptor elements. GRK2 phosphorylates only the C-terminal Ser residues, whereas GRK5 phosphorylates Ser and Thr residues located in intracellular loop 3 and the C-terminus. Interestingly, phosphorylation assays using a series of NTSR1 mutants show that GRK2 does not require acidic residues upstream of the phospho-acceptors for site-specific phosphorylation, in contrast to the β2-adrenergic and μ-opioid receptors. Differential phosphorylation of GPCRs by GRKs is thought to encode a particular signaling outcome, and our in vitro study revealed NTSR1 differential phosphorylation by GRK2 and GRK5.
PMCID: PMC4512254  PMID: 26120872
22.  Crystal Structure of Schistosoma mansoni Arginase, a Potential Drug Target for the Treatment of Schistosomiasis 
Biochemistry  2014;53(28):4671-4684.
The X-ray crystal structure of arginase from Schistosoma mansoni (SmARG) and the structures of its complexes with several amino acid inhibitors have been determined at atomic resolution. SmARG is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to form l-ornithine and urea, and this enzyme is upregulated in all forms of the parasite that interact with the human host. Current hypotheses suggest that parasitic arginases could play a role in host immune evasion by depleting pools of substrate l-arginine that would otherwise be utilized for NO biosynthesis and NO-dependent processes in the immune response. Although SmARG shares only 42% overall amino acid sequence identity with human arginase I, residues important for substrate binding and catalysis are strictly conserved. In general, classical amino acid inhibitors such as 2(S)-amino-6-boronohexanoic acid (ABH) tend to bind more weakly to SmARG than to human arginase I despite identical inhibitor binding modes in each enzyme active site. The identification of a patch on the enzyme surface capable of accommodating the additional Cα-substitutent of an α,α-disubstituted amino acid inhibitor suggests that such inhibitors could exhibit higher affinity and biological activity. The structures of SmARG complexed with two different α,α-disubstituted derivatives of ABH are presented and provide proof-of-concept for this approach in the enhancement of enzyme-inhibitor affinity.
PMCID: PMC4138072  PMID: 25007099
23.  Kinetic and Structural Characterization of Tunnel-Perturbing Mutants in Bradyrhizobium japonicum Proline Utilization A 
Biochemistry  2014;53(31):5150-5161.
Proline utilization A from Bradyrhizobium japonicum (BjPutA) is a bifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate Δ1-pyrroline-5-carboxylate/glutamate-γ-semialdehyde (P5C/GSA). In this work, the structure of the channel was explored by inserting large side chain residues at four positions along the channel in BjPutA. Kinetic analysis of the different mutants revealed replacement of D779 with Tyr (D779Y) or Trp (D779W) significantly decreased the overall rate of the PRODH–P5CDH channeling reaction. X-ray crystal structures of D779Y and D779W revealed that the large side chains caused a constriction in the central section of the tunnel, thus likely impeding the travel of P5C/GSA in the channel. The D779Y and D779W mutants have PRODH activity similar to that of wild-type BjPutA but exhibit significantly lower P5CDH activity, suggesting that exogenous P5C/GSA enters the channel upstream of Asp779. Replacement of nearby Asp778 with Tyr (D778Y) did not impact BjPutA channeling activity. Consistent with the kinetic results, the X-ray crystal structure of D778Y shows that the main channel pathway is not impacted; however, an off-cavity pathway is closed off from the channel. These findings provide evidence that the off-cavity pathway is not essential for substrate channeling in BjPutA.
PMCID: PMC4131897  PMID: 25046425
24.  Mechanistic Insights into the Allosteric Modulation of Opioid Receptors by Sodium Ions 
Biochemistry  2014;53(31):5140-5149.
The idea of sodium ions altering G-protein-coupled receptor (GPCR) ligand binding and signaling was first suggested for opioid receptors (ORs) in the 1970s and subsequently extended to other GPCRs. Recently published ultra-high-resolution crystal structures of GPCRs, including that of the δ-OR subtype, have started to shed light on the mechanism underlying sodium control in GPCR signaling by revealing details of the sodium binding site. Whether sodium accesses different receptor subtypes from the extra- or intracellular sides, following similar or different pathways, is still an open question. Earlier experiments in brain homogenates suggested a differential sodium regulation of ligand binding to the three major OR subtypes, in spite of their high degree of sequence similarity. Intrigued by this possibility, we explored the dynamic nature of sodium binding to δ-OR, μ-OR, and κ-OR by means of microsecond-scale, all-atom molecular dynamics (MD) simulations. Rapid sodium permeation was observed exclusively from the extracellular milieu, and following similar binding pathways in all three ligand-free OR systems, notwithstanding extra densities of sodium observed near nonconserved residues of κ-OR and δ-OR, but not in μ-OR. We speculate that these differences may be responsible for the differential increase in antagonist binding affinity of μ-OR by sodium resulting from specific ligand binding experiments in transfected cells. On the other hand, sodium reduced the level of binding of subtype-specific agonists to all OR subtypes. Additional biased and unbiased MD simulations were conducted using the δ-OR ultra-high-resolution crystal structure as a model system to provide a mechanistic explanation for this experimental observation.
PMCID: PMC4131901  PMID: 25073009
25.  Electron Paramagnetic Resonance and Electron-Nuclear Double Resonance Studies of the Reactions of Cryogenerated Hydroperoxoferric–Hemoprotein Intermediates 
Biochemistry  2014;53(30):4894-4903.
The fleeting ferric peroxo and hydroperoxo intermediates of dioxygen activation by hemoproteins can be readily trapped and characterized during cryoradiolytic reduction of ferrous hemoprotein–O2 complexes at 77 K. Previous cryoannealing studies suggested that the relaxation of cryogenerated hydroperoxoferric intermediates of myoglobin (Mb), hemoglobin, and horseradish peroxidase (HRP), either trapped directly at 77 K or generated by cryoannealing of a trapped peroxo-ferric state, proceeds through dissociation of bound H2O2 and formation of the ferric heme without formation of the ferryl porphyrin π-cation radical intermediate, compound I (Cpd I). Herein we have reinvestigated the mechanism of decays of the cryogenerated hydroperoxyferric intermediates of α- and β-chains of human hemoglobin, HRP, and chloroperoxidase (CPO). The latter two proteins are well-known to form spectroscopically detectable quasistable Cpds I. Peroxoferric intermediates are trapped during 77 K cryoreduction of oxy Mb, α-chains, and β-chains of human hemoglobin and CPO. They convert into hydroperoxoferric intermediates during annealing at temperatures above 160 K. The hydroperoxoferric intermediate of HRP is trapped directly at 77 K. All studied hydroperoxoferric intermediates decay with measurable rates at temperatures above 170 K with appreciable solvent kinetic isotope effects. The hydroperoxoferric intermediate of β-chains converts to the S = 3/2 Cpd I, which in turn decays to an electron paramagnetic resonance (EPR)-silent product at temperature above 220 K. For all the other hemoproteins studied, cryoannealing of the hydroperoxo intermediate directly yields an EPR-silent majority product. In each case, a second follow-up 77 K γ-irradiation of the annealed samples yields low-spin EPR signals characteristic of cryoreduced ferrylheme (compound II, Cpd II). This indicates that in general the hydroperoxoferric intermediates relax to Cpd I during cryoanealing at low temperatures, but when this state is not captured by reaction with a bound substrate, it is reduced to Cpd II by redox-active products of radiolysis.
PMCID: PMC4144713  PMID: 25046203

Results 1-25 (4793)