Biomedical subjects

William R Montfort

Publications and source records attributed to William R Montfort.

15 recordsLinked to original sources

PubMed2006

Structure of the Y94F mutant of Escherichia coli thymidylate synthase.

Tyr94 of Escherichia coli thymidylate synthase is thought to be involved, either directly or by activation of a water molecule, in the abstraction of a proton from C5 of the 2'-deoxyuridine 5'-monophosphate (dUMP) substrate. Mutation of Tyr94 leads to a 400-fold loss in catalytic activity. The structure of the Y94F mutant has been determined in the native state and as a ternary complex with thymidine 5'-monophosphate (dTMP) and 10-propargyl 5,8-dideazafolate (PDDF). There are no structural changes ascribable to the mutation other than loss of a water molecule hydrogen bonded to the tyrosine OH, which is consistent with a catalytic role for the phenolic OH.

Amino Acid Substitution↗

PubMed2006

Crystal structures of thymidylate synthase mutant R166Q: structural basis for the nearly complete loss of catalytic activity.

Thymidylate synthase (TS) catalyzes the folate-dependent methylation of deoxyuridine monophosphate (dUMP) to form thymidine monophosphate (dTMP). We have investigated the role of invariant arginine 166, one of four arginines that contact the dUMP phosphate, using site-directed mutagenesis, X-ray crystallography, and TS from Escherichia coli. The R166Q mutant was crystallized in the presence of dUMP and a structure determined to 2.9 A resolution, but neither the ligand nor the sulfate from the crystallization buffer was found in the active site. A second structure determined with crystals prepared in the presence of dUMP and the antifolate 10-propargyl-5,8-dideazafolate revealed that the inhibitor was bound in an extended, nonproductive conformation, partially occupying the nucleotide-binding site. A sulfate ion, rather than dUMP, was found in the nucleotide phosphate-binding site. Previous studies have shown that the substitution at three of the four arginines of the dUMP phosphate-binding site is permissive; however; for Arg166, all the mutations lead to a near-inactive mutant. The present structures of TS R166Q reveal that the phosphate-binding site is largely intact, but with a substantially reduced affinity for phosphate, despite the presence of the three remaining arginines. The position of Cys146, which initiates catalysis, is shifted in the mutant and resides in a position that interferes with the binding of the dUMP pyrimidine moiety.

Amino Acid Substitution↗

PubMed2005

Ultrahigh resolution structures of nitrophorin 4: heme distortion in ferrous CO and NO complexes.

Nitrophorin 4 (NP4), a nitric oxide (NO)-transport protein from the blood-sucking insect Rhodnius prolixus, uses a ferric (Fe3+) heme to deliver NO to its victims. NO binding to NP4 induces a large conformational change and complete desolvation of the distal pocket. The heme is markedly nonplanar, displaying a ruffling distortion postulated to contribute to stabilization of the ferric iron. Here, we report the ferrous (Fe2+) complexes of NP4 with NO, CO, and H2O formed after chemical reduction of the protein and the characterization of these complexes by absorption spectroscopy, flash photolysis, and ultrahigh-resolution crystallography (resolutions vary from 0.9 to 1.08 A). The absorption spectra, both in solution and in the crystal, are typical for six-coordinated ferrous complexes. Closure and desolvation of the distal pocket occurs upon binding CO or NO to the iron regardless of the heme oxidation state, confirming that the conformational change is driven by distal ligand polarity. The degree of heme ruffling is coupled to the nature of the ligand and the iron oxidation state in the following order: (Fe3+)-NO > (Fe2+)-NO > (Fe2+)-CO > (Fe3+)-H2O > (Fe2+)-H2O. The ferrous coordination geometry is as expected, except for the proximal histidine bond, which is shorter than typically found in model compounds. These data are consistent with heme ruffling and coordination geometry serving to stabilize the ferric state of the nitrophorins, a requirement for their physiological function. Possible roles for heme distortion and NO bending in heme protein function are discussed.

Carbon Monoxide↗

PubMed2005

A novel copper-binding fold for the periplasmic copper resistance protein CusF.

We have determined the crystal structure of apo-CusF, a periplasmic protein involved in copper and silver resistance in Escherichia coli. The protein forms a five-stranded beta-barrel, classified as an OB-fold, which is a unique topology for a copper-binding protein. NMR chemical shift mapping experiments suggest that Cu(I) is bound by conserved residues H36, M47, and M49 located in beta-strands 2 and 3. These residues are clustered at one end of the beta-barrel, and their side chains are oriented toward the interior of the barrel. Cu(I) can be modeled into the apo-CusF structure with only minimal structural changes using H36, M47, and M49 as ligands. The unique structure and metal binding site of CusF are distinct from those of previously characterized copper-binding proteins.

Amino Acid Sequence↗

PubMed2005

Heme-assisted S-nitrosation of a proximal thiolate in a nitric oxide transport protein.

Certain bloodsucking insects deliver nitric oxide (NO) while feeding, to induce vasodilation and inhibit blood coagulation. We have expressed, characterized, and determined the crystal structure of the Cimex lectularius (bedbug) nitrophorin, the protein responsible for NO storage and delivery, to understand how the insect successfully handles this reactive molecule. Surprisingly, NO binds not only to the ferric nitrophorin heme, but it can also be stored as an S-nitroso (SNO) conjugate of the proximal heme cysteine (Cys-60) when present at higher concentrations. EPR- and UV-visible spectroscopies, and a crystallographic structure determination to 1.75-A resolution, reveal SNO formation to proceed with reduction of the heme iron, yielding an Fe-NO complex. Stopped-flow kinetic measurements indicate that an ordered reaction mechanism takes place: initial NO binding occurs at the ferric heme and is followed by heme reduction, Cys-60 release from the heme iron, and SNO formation. Release of NO occurs through a reversal of these steps. These data provide, to our knowledge, the first view of reversible metal-assisted SNO formation in a protein and suggest a mechanism for its role in NO release from ferrous heme. This mechanism and Cimex nitrophorin structure are completely unlike those of the nitrophorins from Rhodnius prolixus, where NO protection is provided by a large conformational change that buries the heme nitrosyl complex, highlighting the remarkable evolution of proteins that assist insects in bloodfeeding.

Animals↗

PubMed2005

5,10-Methylene-5,6,7,8-tetrahydrofolate conformational transitions upon binding to thymidylate synthase: molecular mechanics and continuum solvent studies.

We applied the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach to evaluate relative stability of the extended (flat) and C-shaped (bent) solution conformational forms of the 5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) molecule in aqueous solution. Calculations indicated that both forms have similar free energies in aqueous solution but detailed energy components are different. The bent solution form has lower intramolecular electrostatic and van der Waals interaction energies. The flat form has more favorable solvation free energy and lower contribution from the bond, angle and torsion angle molecular mechanical internal energies. We exploit these results and combine them with known crystallographic data to provide a model for the progressive binding of the mTHF molecule, a natural cofactor of thymidylate synthase (TS), to the complex forming in the TS-catalyzed reaction. We propose that at the time of initial weak binding in the open enzyme the cofactor molecule remains in a close balance between the flat and bent solution conformations, with neither form clearly favored. Later, thymidylate synthase undergoes conformational change leading to the closure of the active site and the mTHF molecule is withdrawn from the solvent. That effect shifts the thermodynamic equilibrium of the mTHF molecule toward the bent solution form. At the same time, burying the cofactor molecule in the closed active site produces numerous contacts between mTHF and protein that render change in the shape of the mTHF molecule. As a result, the bent solution conformer is converted to more strained L-shaped bent enzyme conformer of the mTHF molecule. The strain in the bent enzyme conformation allows for the tight binding of the cofactor molecule to the productive ternary complex that forms in the closed active site, and facilitates the protonation of the imidazolidine N10 atom, which promotes further reaction.

Hydrogen Bonding↗

PubMed2004

Protein functional cycle viewed at atomic resolution: conformational change and mobility in nitrophorin 4 as a function of pH and NO binding.

The blood-sucking insect Rhodnius prolixus uses nitrophorin 4, a heme protein, to deliver nitric oxide (NO) to a victim, causing vasodilation and improved feeding. Binding of NO occurs at a ferric heme and is modulated by pH. NO binding at lower pH induces a large conformational change involving loops A-B and G-H that leads to distal pocket desolvation and protection of the nitrosyl heme complex. We have determined the crystal structures of Rhodnius nitrophorin 4 to ultrahigh resolution in four functional states: +/-NO at pH = 7.4 and +/-NO at pH = 5.6. The structure with NO at pH 7.4 (1.08 A) is newly determined while the other complexes have been modeled to resolutions much greater than previously reported (1.0-0.85 A). The ultrahigh resolution allowed us to resolve multiple conformers in binding-site loops, leading to a detailed description of the dynamics involved with storing NO in the insect salivary gland at low pH, and releasing NO in response to the increased pH of a victim's tissue. Strikingly, features for both the "open" and "closed" conformers exist under all conditions, suggesting that the flexible loops can transition with relative ease between conformational states. Yet, release of NO from rNP4 is much slower than found for other ferric heme proteins. The structures suggest that highly mobile loops can limit diffusion of diatomic molecules into and out of a protein cavity, a result with implications for the role of protein dynamics in function.

Animals↗

PubMed2004

Structural dynamics controls nitric oxide affinity in nitrophorin 4.

Nitrophorin 4 (NP4) is one of seven nitric oxide (NO) transporting proteins in the blood-sucking insect Rhodnius prolixus. In its physiological function, NO binds to a ferric iron centered in a highly ruffled heme plane. Carbon monoxide (CO) also binds after reduction of the heme iron. Here we have used Fourier transform infrared spectroscopy at cryogenic temperatures to study CO and NO binding and migration in NP4, complemented by x-ray cryo-crystallography on xenon-containing NP4 crystals to identify cavities that may serve as ligand docking sites. Multiple infrared stretching bands of the heme-bound ligands indicate different active site conformations with varying degrees of hydrophobicity. Narrow infrared stretching bands are observed for photodissociated CO and NO; temperature-derivative spectroscopy shows that these bands are associated with ligand docking sites close to the extremely reactive heme iron. No rebinding from distinct secondary sites was detected, although two xenon binding cavities were observed in the x-ray structure. Photolysis studies at approximately 200 K show efficient NO photoproduct formation in the more hydrophilic, open NP4 conformation. This result suggests that ligand escape is facilitated in this conformation, and blockage of the active site by water hinders immediate reassociation of NO to the ferric iron. In the closed, low-pH conformation, ligand escape from the active site of NP4 is prevented by an extremely reactive heme iron and the absence of secondary ligand docking sites.

Animals↗

PubMed2004

Role of binding site loops in controlling nitric oxide release: structure and kinetics of mutant forms of nitrophorin 4.

Nitrophorins are ferric heme proteins that transport nitric oxide (NO) from blood-sucking insects to victims. NO binding is tighter at lower pH values, as found in the insect salivary gland, and weaker at the pH of the victim's tissue, facilitating NO release and subsequent vasodilation. Previous structural analyses of nitrophorin 4 (NP4) from Rhodnius prolixus revealed a substantial NO-induced conformational change involving the A-B and G-H loops, which rearrange to desolvate the distal pocket and pack nonpolar residues against the heme-ligated NO. Previous kinetic analyses revealed a slow, biphasic, and pH-dependent NO release, which was proposed to be associated with loop movements. In this study, we created NP4 mutants D30A and D30N (A-B loop), D129A/L130A (G-H loop), and T121V (distal pocket). Eight crystal structures were determined, including complexes with NO, NH(3), and imidazole, to resolutions as high as 1.0 A. The NO-induced conformational change is largely abolished in the loop mutants, but retained in T121V. Kinetic analyses using stopped-flow spectroscopy revealed the pH dependence for NO release is eliminated for D129A/L130A, considerably reduced for D30A and D30N, but retained for T121V. NO association rates were increased 2-5-fold for T121V, but were unchanged in the loop mutants. Taken together, our findings demonstrate that the pH dependency for NO release is linked to loop dynamics and that solvent reorganization is apparently rate-limiting for formation of the initial iron-nitrosyl bond. Interestingly, the multiphasic kinetic behavior of rNPs was not affected by mutations, and its cause remains unclear.

Binding Sites↗

PubMed2004

Cuprous oxidase activity of CueO from Escherichia coli.

We have found CueO from Escherichia coli to have a robust cuprous oxidase activity, severalfold higher than any homologue. These data suggest that a functional role for CueO in protecting against copper toxicity in vivo includes the removal of Cu(I).

Copper↗

PubMed2003

Axial ligand complexes of the Rhodnius nitrophorins: reduction potentials, binding constants, EPR spectra, and structures of the 4-iodopyrazole and imidazole complexes of NP4.

Previously, we utilized 4-iodopyrazole (4IPzH) as a heavy atom derivative for the initial solution of the crystal structure of the nitrophorin from Rhodnius prolixus, NP1, where it was found to bind to the heme with the iodo group disordered in two positions. We have now determined the structure of the 4IPzH complex of NP4 at pH 7.5 and find that the geometry and bond lengths at the iron center are extremely similar to those of the imidazole (ImH) complex of the same protein (structure determined at pH 5.6), except that the G-H loop is not in the closed conformation. 4IPzH binds to the heme of NP4 in an ordered manner, with the iodo substituent pointed toward the opening of the heme pocket, near the surface of the protein. In order to understand the solution chemistry in terms of the relative binding abilities of 4IPzH, ImH, and histamine (Hm, a physiological ligand for the nitrophorins), we have also investigated the equilibrium binding constants and reduction potentials of these three ligand complexes of the four Rhodnius nitrophorins as a function of pH. We have found that, unlike the other Lewis bases, 4IPzH forms less stable complexes with the Fe(III) than the Fe(II) oxidation states of NP1 and NP4, and similar stability for the two oxidation states of NP2 and NP3, suggesting that this ligand is a softer base than ImH or Hm, for both of which the Fe(III) complexes are more stable than those of Fe(II) for all four nitrophorins. Surprisingly, in spite of this and the much lower basicity of 4IPzH than imidazole and histamine, the EPR g-values of all three ligand complexes are very similar.

Algorithms↗

PubMed2003

Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif.

Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional non-active site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E0) of the Trx1 active site (-230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an alpha helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the non-active site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1 activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals.

Amino Acid Motifs↗

PubMed2003

A labile regulatory copper ion lies near the T1 copper site in the multicopper oxidase CueO.

CueO, a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli, is expressed under conditions of copper stress and shows enhanced oxidase activity when additional copper is present. The 1.7-A resolution structure of a crystal soaked in CuCl2 reveals a Cu(II) ion bound to the protein 7.5 A from the T1 copper site in a region rich in methionine residues. The trigonal bipyramidal coordination sphere is unusual, containing two methionine sulfur atoms, two aspartate carboxylate oxygen atoms, and a water molecule. Asp-439 both ligates the labile copper and hydrogen-bonds to His-443, which ligates the T1 copper. This arrangement may mediate electron transfer from substrates to the T1 copper. Mutation of residues bound to the labile copper results in loss of oxidase activity and of copper tolerance, confirming a regulatory role for this site. The methionine-rich portion of the protein, which is similar to that of other proteins involved in copper homeostasis, does not display additional copper binding. The type 3 copper atoms of the trinuclear cluster in the structure are bridged by a chloride ion that completes a square planar coordination sphere for the T2 copper atom but does not affect oxidase activity.

Base Sequence↗

PubMed2003

Complementing thymidylate synthase.

The structure of thymidylate synthase complementing protein with substrates dUMP and FAD, presented in this issue of Structure, sheds light on a fascinating new catalytic mechanism, suggests a strategy for the design of new antimicrobial compounds, and highlights the promise of proteomics in medicine.

Deoxyuracil Nucleotides↗

PubMed2002

Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli.

CueO (YacK), a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli. The crystal structure of CueO has been determined to 1.4-A resolution by using multiple anomalous dispersion phasing and an automated building procedure that yielded a nearly complete model without manual intervention. This is the highest resolution multicopper oxidase structure yet determined and provides a particularly clear view of the four coppers at the catalytic center. The overall structure is similar to those of laccase and ascorbate oxidase, but contains an extra 42-residue insert in domain 3 that includes 14 methionines, nine of which lie in a helix that covers the entrance to the type I (T1, blue) copper site. The trinuclear copper cluster has a conformation not previously seen: the Cu-O-Cu binuclear species is nearly linear (Cu-O-Cu bond angle = 170 degrees) and the third (type II) copper lies only 3.1 A from the bridging oxygen. CueO activity was maximal at pH 6.5 and in the presence of >100 microM Cu(II). Measurements of intermolecular and intramolecular electron transfer with laser flash photolysis in the absence of Cu(II) show that, in addition to the normal reduction of the T1 copper, which occurs with a slow rate (k = 4 x 10(7) M(-1)x (-1)), a second electron transfer process occurs to an unknown site, possibly the trinuclear cluster, with k = 9 x 10(7) M(-1) x (-1), followed by a slow intramolecular electron transfer to T1 copper (k approximately 10 s(-1)). These results suggest the methionine-rich helix blocks access to the T1 site in the absence of excess copper.

Copper↗