Cancer-associated somatic mutations in human phosphofructokinase-1 reveal a critical electrostatic interaction for allosteric regulation of enzyme activity.

Metabolic reprogramming, including increased glucose uptake and lactic acid excretion, is a hallmark of cancer. The glycolytic 'gatekeeper' enzyme phosphofructokinase-1 (PFK1), which catalyzes the step committing glucose to breakdown, is dysregulated in cancers. While altered PFK1 activity and expression in tumors have been demonstrated, little is known about the effects of cancer-associated somatic mutations. Somatic mutations in PFK1 inform our understanding of allosteric regulation by identifying key amino acid residues involved in the regulation of enzyme activity. Here, we characterized mutations disrupting an evolutionarily conserved salt bridge between aspartic acid and arginine in human platelet (PFKP) and liver (PFKL) isoforms. Using purified recombinant proteins, we showed that disruption of the Asp-Arg pair in two PFK1 isoforms decreased enzyme activity and altered allosteric regulation. We determined the crystal structure of PFK1 to 3.6 Šresolution and used molecular dynamic simulations to understand molecular mechanisms of altered allosteric regulation. We showed that PFKP-D564N had a decreased total system energy and changes in the electrostatic surface potential of the effector site. Cells expressing PFKP-D564N demonstrated a decreased rate of glycolysis, while their ability to induce glycolytic flux under conditions of low cellular energy was enhanced compared with cells expressing wild-type PFKP. Taken together, these results suggest that mutations in Arg-Asp pair at the interface of the catalytic-regulatory domains stabilizes the t-state and presents novel mechanistic insight for therapeutic development in cancer.

Crystal Structure and Conformational Dynamics of Pyrococcus furiosus Prolyl Oligopeptidase.

Enzymes in the prolyl oligopeptidase family possess unique structures and substrate specificities that are important for their biological activity and for potential biocatalytic applications. The crystal structures of Pyrococcus furiosus ( Pfu) prolyl oligopeptidase (POP) and the corresponding S477C mutant were determined to 1.9 and 2.2 Å resolution, respectively. The wild type enzyme crystallized in an open conformation, indicating that this state is readily accessible, and it contained bound chloride ions and a prolylproline ligand. These structures were used as starting points for molecular dynamics simulations of Pfu POP conformational dynamics. The simulations showed that large-scale domain opening and closing occurred spontaneously, providing facile substrate access to the active site. Movement of the loop containing the catalytically essential histidine into a conformation similar to those found in structures with fully formed catalytic triads also occurred. This movement was modulated by chloride binding, providing a rationale for experimentally observed activation of POP peptidase catalysis by chloride. Thus, the structures and simulations reported in this study, combined with existing biochemical data, provide a number of insights into POP catalysis.

The sole tryptophan of amicyanin enhances its thermal stability but does not influence the electronic properties of the type 1 copper site.

The cupredoxin amicyanin possesses a single tryptophan residue, Trp45. Its fluorescence is quenched when copper is bound even though it is separated by 10.1Å. Mutation of Trp45 to Ala, Phe, Leu and Lys resulted in undetectable protein expression. A W45Y amicyanin variant was isolated. The W45Y mutation did not alter the spectroscopic properties or intrinsic redox potential of amicyanin, but increased the pKa value for the pH-dependent redox potential by 0.5 units. This is due to a hydrogen-bond involving the His95 copper ligand which is present in reduced W45Y amicyanin but not in native amicyanin. The W45Y mutation significantly decreased the thermal stability of amicyanin, as determined by changes in the visible absorbance of oxidized amicyanin and in the circular dichroism spectra for oxidized, reduced and apo forms of amicyanin. Comparison of the crystal structures suggests that the decreased stability of W45Y amicyanin may be attributed to the loss of a strong interior hydrogen bond between Trp45 and Tyr90 in native amicyanin which links two of the ß-sheets that comprise the overall structure of amicyanin. Thus, Trp45 is critical for stabilizing the structure of amicyanin but it does not influence the electronic properties of the copper which quenches its fluorescence.

Analysis of Laboratory-Evolved Flavin-Dependent Halogenases Affords a Computational Model for Predicting Halogenase Site Selectivity.

Flavin-dependent halogenases (FDHs) catalyze selective halogenation of electron-rich aromatic compounds without the need for harsh oxidants required by conventional oxidative halogenation reactions. Predictive models for halogenase site selectivity could greatly improve their utility for chemical synthesis. Toward this end, we analyzed the structures and selectivity of three halogenase variants evolved to halogenate tryptamine with orthogonal selectivity. Crystal structures and reversion mutations revealed key residues involved in altering halogenase selectivity. Density functional theory calculations and molecular dynamics simulations are both consistent with hypohalous acid as the active halogenating species in FDH catalysis. This model was used to accurately predict the site selectivity of halogenase variants toward different synthetic substrates, providing a valuable tool for implementing halogenases in biocatalysis efforts.

Proline 96 of the copper ligand loop of amicyanin regulates electron transfer from methylamine dehydrogenase by positioning other residues at the protein-protein interface.

Amicyanin is a type 1 copper protein that serves as an electron acceptor for methylamine dehydrogenase (MADH). The site of interaction with MADH is a "hydrophobic patch" of amino acid residues including those that comprise a "ligand loop" that provides three of the four copper ligands. Three prolines are present in this region. Pro94 of the ligand loop was previously shown to strongly influence the redox potential of amicyanin but not affinity for MADH or mechanism of electron transfer (ET). In this study Pro96 of the ligand loop was mutated. P96A and P96G mutations did not affect the spectroscopic or redox properties of amicyanin but increased the K(d) for complex formation with MADH and altered the kinetic mechanism for the interprotein ET reaction. Values of reorganization energy (λ) and electronic coupling (H(AB)) for the ET reaction with MADH were both increased by the mutation, indicating that the true ET reaction observed with native amicyanin was now gated by or coupled to a reconfiguration of the proteins within the complex. The crystal structure of P96G amicyanin was very similar to that of native amicyanin, but notably, in addition to the change in Pro96, the side chains of residues Phe97 and Arg99 were oriented differently. These two residues were previously shown to make contacts with MADH that were important for stabilizing the amicyanin-MADH complex. The values of K(d), λ, and H(AB) for the reactions of the Pro96 mutants with MADH are remarkably similar to those obtained previously for P52G amicyanin. Mutation of this proline, also in the hydrophobic patch, caused reorientation of the side chain of Met51, another reside that interacted with MADH and caused a change in the kinetic mechanism of ET from MADH. These results show that proline residues near the copper site play key roles in positioning other amino acid residues at the amicyanin-MADH interface not only for specific binding to the redox protein partner but also to optimize the orientation of proteins for interprotein ET.

Structural basis of antagonizing the vitamin K catalytic cycle for anticoagulation.

Vitamin K antagonists are widely used anticoagulants that target vitamin K epoxide reductases (VKOR), a family of integral membrane enzymes. To elucidate their catalytic cycle and inhibitory mechanism, we report 11 x-ray crystal structures of human VKOR and pufferfish VKOR-like, with substrates and antagonists in different redox states. Substrates entering the active site in a partially oxidized state form cysteine adducts that induce an open-to-closed conformational change, triggering reduction. Binding and catalysis are facilitated by hydrogen-bonding interactions in a hydrophobic pocket. The antagonists bind specifically to the same hydrogen-bonding residues and induce a similar closed conformation. Thus, vitamin K antagonists act through mimicking the key interactions and conformational changes required for the VKOR catalytic cycle.

Preliminary neutron and X-ray crystallographic studies of equine cyanomethemoglobin.

Room-temperature and 100 K X-ray and room-temperature neutron diffraction data have been measured from equine cyanomethemoglobin to 1.7 A resolution using a home source, to 1.6 A resolution on NE-CAT at the Advanced Photon Source and to 2.0 A resolution on the PCS at Los Alamos Neutron Science Center, respectively. The cyanomethemoglobin is in the R state and preliminary room-temperature electron and neutron scattering density maps clearly show the protonation states of potential Bohr groups. Interestingly, a water molecule that is in the vicinity of the heme group and coordinated to the distal histidine appears to be expelled from this site in the low-temperature structure.

Defining the role of the axial ligand of the type 1 copper site in amicyanin by replacement of methionine with leucine.

The effects of replacing the axial methionine ligand of the type 1 copper site with leucine on the structure and function of amicyanin have been characterized. The crystal structures of the oxidized and reduced forms of the protein reveal that the copper site is now tricoordinate with no axial ligand, and that the copper coordination distances for the two ligands provided by histidines are significantly increased. Despite these structural changes, the absorption and EPR spectra of M98L amicyanin are only slightly altered and still consistent with that of a typical type 1 site. The oxidation-reduction midpoint potential (E(m)) value becomes 127 mV more positive as a consequence of the M98L mutation, most likely because of the increased hydrophobicity of the copper site. The most dramatic effect of the mutation was on the electron transfer (ET) reaction from reduced M98L amicyanin to cytochrome c(551i) within the protein ET complex. The rate decreased 435-fold, which was much more than expected from the change in E(m). Examination of the temperature dependence of the ET rate (k(ET)) revealed that the mutation caused a 13.6-fold decrease in the electronic coupling (H(AB)) for the reaction. A similar decrease was predicted from a comparative analysis of the crystal structures of reduced M98L and native amicyanins. The most direct route of ET for this reaction is through the Met98 ligand. Inspection of the structures suggests that the major determinant of the large decrease in the experimentally determined values of H(AB) and k(ET) is the increased distance from the copper to the protein within the type 1 site of M98L amicyanin.

Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates.

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful. However, the G81A mutants of MDH and of MDH-GOX2 displayed approximately 100-fold lower reactivity with substrate and a modestly higher reactivity towards molecular oxygen. In order to understand the effect of the mutation and to identify the mode of substrate binding in MDH-GOX2, a crystallographic investigation of the G81A mutant of the MDH-GOX2 enzyme was initiated. The structures of ligand-free G81A mutant MDH-GOX2 and of its complexes with the substrates 2-hydroxyoctanoate and 2-hydroxy-3-indolelactate were determined at 1.6, 2.5 and 2.2 A resolution, respectively. In the ligand-free G81A mutant protein, a sulfate anion previously found at the active site is displaced by the alanine side chain introduced by the mutation. 2-Hydroxyoctanoate binds in an apparently productive mode for subsequent reaction, while 2-hydroxy-3-indolelactate is bound to the enzyme in an apparently unproductive mode. The results of this investigation suggest that a lowering of the polarity of the flavin environment resulting from the displacement of nearby water molecules caused by the glycine-to-alanine mutation may account for the lowered catalytic activity of the mutant enzyme, which is consistent with the 30 mV lower flavin redox potential. Furthermore, the altered binding mode of the indolelactate substrate may account for its reduced activity compared with octanoate, as observed in the crystalline state.

Crystallographic studies on B12 binding proteins in eukaryotes and prokaryotes.

The X-ray crystal structures of several important vitamin B12 binding proteins that have been solved in recent years have enhanced our current understanding in the vitamin B12 field. These structurally diverse groups of B12 binding proteins perform various important biological activities, both by transporting B12 as well as catalyzing various biological reactions. An in-depth comparative analysis of these structures was carried out using PDB coordinates of a carefully chosen database of B12 binding proteins to correlate the overall folding of the molecule with phylogeny, the B12 interactions, and with their biological function. The structures of these proteins are discussed in the context of this comparative analysis.

Replacement of the axial copper ligand methionine with lysine in amicyanin converts it to a zinc-binding protein that no longer binds copper.

The mutation of the axial ligand of the type I copper protein amicyanin from Met to Lys results in a protein that is spectroscopically invisible and redox inactive. M98K amicyanin acts as a competitive inhibitor in the reaction of native amicyanin with methylamine dehydrogenase indicating that the M98K mutation has not affected the affinity for its natural electron donor. The crystal structure of M98K amicyanin reveals that its overall structure is very similar to native amicyanin but that the type I binding site is occupied by zinc. Anomalous difference Fourier maps calculated using the data collected around the absorption edges of copper and zinc confirm the presence of Zn(2+) at the type I site. The Lys98 NZ donates a hydrogen bond to a well-ordered water molecule at the type I site which enhances the ability of Lys98 to provide a ligand for Zn(2+). Attempts to reconstitute M98K apoamicyanin with copper resulted in precipitation of the protein. The fact that the M98K mutation generated such a selective zinc-binding protein was surprising as ligation of zinc by Lys is rare and this ligand set is unique for zinc.

Crystal structure of an electron transfer complex between aromatic amine dehydrogenase and azurin from Alcaligenes faecalis.

The crystal structure of an electron transfer complex of aromatic amine dehydrogenase (AADH) and azurin is presented. Electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of AADH to the type I copper of the cupredoxin azurin. This structure is compared with the complex of the TTQ-containing methylamine dehydrogenase (MADH) and the cupredoxin amicyanin. Despite significant similarities between the two quinoproteins and the two cupredoxins, each is specific for its respective partner and the ionic strength dependence and magnitude of the binding constant for each complex are quite different. The AADH-azurin interface is largely hydrophobic, covering approximately 500 A(2) of surface on each molecule, with one direct hydrogen bond linking them. The closest distance from TTQ to copper is 12.6 A compared with a distance of 9.3 A in the MADH-amicyanin complex. When the MADH-amicyanin complex is aligned with the AADH-azurin complex, the amicyanin lies on top of the azurin but is oriented quite differently. Although the copper atoms differ in position by approximately 4.7 A, the amicyanin bound to MADH appears to be rotated approximately 90 degrees from its aligned position with azurin. Comparison of the structures of the two complexes identifies features of the interface that dictate the specificity of the protein-protein interaction and determine the rate of interprotein electron transfer.

A novel, potent dual inhibitor of the leukocyte proteases cathepsin G and chymase: molecular mechanisms and anti-inflammatory activity in vivo.

Certain leukocytes release serine proteases that sustain inflammatory processes and cause disease conditions, such as asthma and chronic obstructive pulmonary disease. We identified beta-ketophosphonate 1 (JNJ-10311795; RWJ-355871) as a novel, potent dual inhibitor of neutrophil cathepsin G (K(i) = 38 nm) and mast cell chymase (K(i) = 2.3 nm). The x-ray crystal structures of 1 complexed with human cathepsin G (1.85 A) and human chymase (1.90 A) reveal the molecular basis of the dual inhibition. Ligand 1 occupies the S(1) and S(2) subsites of cathepsin G and chymase similarly, with the 2-naphthyl in S(1), the 1-naphthyl in S(2), and the phosphonate group in a complex network of hydrogen bonds. Surprisingly, however, the carboxamido-N-(naphthalene-2-carboxyl)piperidine group is found to bind in two distinct conformations. In cathepsin G, this group occupies the hydrophobic S(3)/S(4) subsites, whereas in chymase, it does not; rather, it folds onto the 1-naphthyl group of the inhibitor itself. Compound 1 exhibited noteworthy anti-inflammatory activity in rats for glycogen-induced peritonitis and lipopolysaccharide-induced airway inflammation. In addition to a marked reduction in neutrophil influx, 1 reversed increases in inflammatory mediators interleukin-1alpha, interleukin-1beta, tissue necrosis factor-alpha, and monocyte chemotactic protein-1 in the glycogen model and reversed increases in airway nitric oxide levels in the lipopolysaccharide model. These findings demonstrate that it is possible to inhibit both cathepsin G and chymase with a single molecule and suggest an exciting opportunity in the treatment of asthma and chronic obstructive pulmonary disease.

High resolution structures of an oxidized and reduced flavoprotein. The water switch in a soluble form of (S)-mandelate dehydrogenase.

The crystal structures of a soluble mutant of the flavoenzyme mandelate dehydrogenase (MDH) from Pseudomonas putida and of the substrate-reduced enzyme have been analyzed at 1.35-A resolution. The mutant (MDH-GOX2) is a fully active chimeric enzyme in which residues 177-215 of the membrane-bound MDH are replaced by residues 176-195 of glycolate oxidase from spinach. Both structures permit full tracing of the polypeptide backbone chain from residues 4-356, including a 4-residue segment that was disordered in an earlier study of the oxidized protein at 2.15 A resolution. The structures of MDH-GOX2 in the oxidized and reduced states are virtually identical with only a slight increase in the bending angle of the flavin ring upon reduction. The only other structural changes within the protein interior are a 10 degrees rotation of an active site tyrosine side chain, the loss of an active site water, and a significant movement of six other water molecules in the active site by 0.45 to 0.78 A. Consistent with solution studies, there is no apparent binding of either the substrate, mandelate, or the oxidation product, benzoylformate, to the reduced enzyme. The observed structural changes upon enzyme reduction have been interpreted as a rearrangement of the hydrogen bonding pattern within the active site that results from binding of a proton to the N-5 position of the anionic hydroquinone form of the reduced flavin prosthetic group. Implications for the low oxidase activity of the reduced enzyme are also discussed.