Christin A. Frederick

Geometry of the soluble methane monooxygenase catalytic diiron center in two oxidation states.

BACKGROUND The hydroxylase component of soluble methane monooxygenase (sMMO) contains a dinuclear iron center responsible for the oxidation of methane to methanol. As isolated, the center is in the oxidized, diiron(III) state. The 2.2 A resolution X-ray structure of the oxidized hydroxylase, Hox, from Methylococcus capsulatus (Bath) was previously determined at 4 degrees C. In this structure the two iron atoms are bridged by a glutamate, a hydroxide ion, and an acetate ion, and additionally coordinated to two His residues, three Glu residues, and a water molecule. RESULTS The 1.7 A resolution crystal structures of the sMMO hydroxylase from Methylococcus capsulatus (Bath) in both its oxidized diiron(III), Hox, and dithionite-treated, reduced diiron(II), Hred, oxidation states were determined at -160 degrees C. The structure of the diiron center in Hox differs from that previously reported at 2.2 A resolution and 4 degrees C. Although the hydroxide bridge is retained, the bidentate, bridging ligand assigned as acetate is replaced by a weakly coordinating monoatomic water bridge. In the resulting four-membered Fe(OH)Fe(OH2) ring, the Fe ... Fe distance is shortened from 3.4 A to 3.1 A. In protomer A of Hred, the hydroxide bridge is displaced by an oxygen atom of Glu243, which undergoes a carboxylate shift from its terminal monodentate binding mode in Hox to a mode in which the carboxylate is both monoatomic bridging and bidentate chelating. We therefore conclude that the center has been reduced to the diiron(II) oxidation state. Both iron atoms are coordinated to five ligands and weakly to a sixth water molecule in the resulting structure. The diiron center in protomer B of Hred has the same composition as those in Hox. In both the oxidized and reduced structures, the diiron core is connected through hydrogen bonds involving exogenous species to Thr213 in the active site cavity. CONCLUSIONS The diiron center in Hox can change its exogenous ligand coordination and geometry, a property that could be important in the catalytic cycle of sMMO. In Hred, a carboxylate shift occurs, extruding hydroxide ion and opening coordination sites for reaction with O2 to form the diiron(III) peroxo intermediate, Hperoxo. Residue Thr213 may function in catalysis.

Crystal Structure of the Tandem Phosphatase Domains of RPTP LAR

Most receptor-like protein tyrosine phosphatases (RPTPs) contain two conserved phosphatase domains (D1 and D2) in their intracellular region. The carboxy-terminal D2 domain has little or no catalytic activity. The crystal structure of the tandem D1 and D2 domains of the human RPTP LAR revealed that the tertiary structures of the LAR D1 and D2 domains are very similar to each other, with the exception of conformational differences at two amino acid positions in the D2 domain. Site-directed mutational changes at these positions (Leu-1644-to-Tyr and Glu-1779-to-Asp) conferred a robust PTPase activity to the D2 domain. The catalytic sites of both domains are accessible, in contrast to the dimeric blocked orientation model previously suggested. The relative orientation of the LAR D1 and D2 domains, constrained by a short linker, is stabilized by extensive interdomain interactions, suggesting that this orientation might be favored in solution.

Crystal structures of the methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): implications for substrate gating and component interactions.

The crystal structure of the nonheme iron‐containing hydroxylase component of methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) has been solved in two crystal forms, one of which was refined to 1.7 Å resolution. The enzyme is composed of two copies each of three subunits (α2β2γ2), and all three subunits are almost completely α‐helical, with the exception of two β hairpin structures in the α subunit. The active site of each α subunit contains one dinuclear iron center, housed in a four‐helix bundle. The two iron atoms are octahedrally coordinated by 2 histidine and 4 glutamic acid residues as well as by a bridging hydroxide ion, a terminal water molecule, and at 4°C, a bridging acetate ion, which is replaced at −160°C with a bridging water molecule. Comparison of the results for two crystal forms demonstrates overall conservation and relative orientation of the domain structures. The most prominent structural difference identified between the two crystal forms is in an altered side chain conformation for Leu 110 at the active site cavity. We suggest that this residue serves as one component of a hydrophobic gate controlling access of substrates to and products from the active site. The leucine gate may be responsible for the effect of the B protein component on the reactivity of the reduced hydroxylase with dioxygen. A potential reductase binding site has been assigned based on an analysis of crystal packing in the two forms and corroborated by inhibition studies with a synthetic peptide corresponding to the proposed docking position. Proteins 29:141–152, 1997.

Structural basis for the function and regulation of the receptor protein tyrosine phosphatase CD45.

CD45 is the prototypic member of transmembrane receptor-like protein tyrosine phosphatases (RPTPs) and has essential roles in immune functions. The cytoplasmic region of CD45, like many other RPTPs, contains two homologous protein tyrosine phosphatase domains, active domain 1 (D1) and catalytically impaired domain 2 (D2). Here, we report crystal structure of the cytoplasmic D1D2 segment of human CD45 in native and phosphotyrosyl peptide-bound forms. The tertiary structures of D1 and D2 are very similar, but doubly phosphorylated CD3ζ immunoreceptor tyrosine-based activation motif peptide binds only the D1 active site. The D2 “active site” deviates from the other active sites significantly to the extent that excludes any possibility of catalytic activity. The relative orientation of D1 and D2 is very similar to that observed in leukocyte common antigen–related protein with both active sites in an open conformation and is restrained through an extensive network of hydrophobic interactions, hydrogen bonds, and salt bridges. This crystal structure is incompatible with the wedge model previously suggested for CD45 regulation.

Intramolecular interactions of the regulatory domains of the Bcr-Abl kinase reveal a novel control mechanism

BACKGROUND The Abl nonreceptor tyrosine kinase is implicated in a range of cellular processes and its transforming variants are involved in human leukemias. The N-terminal regulatory region of the Abl protein contains Src homology domains SH2 and SH3 which have been shown to be important for the regulation of its activity in vivo. These domains are often found together in the same protein and biochemical data suggest that the functions of one domain can be influenced by the other. RESULTS We have determined the crystal structure of the Abl regulatory region containing the SH3 and SH2 domains. In general, the individual domains are very similar to those of previously solved structures, although the Abl SH2 domain contains a loop which is extended so that one side of the resulting phosphotyrosine-binding pocket is open. In our structure the protein exists as a monomer with no intermolecular contacts to which a biological function may be attributed. However, there is a significant intramolecular contact between a loop of the SH3 domain and the extended loop of the SH2 domain. This contact surface includes the SH2 loop segment that is responsible for binding the phosphate moiety of phosphotyrosine-containing proteins and is therefore critical for orienting peptide interactions. CONCLUSIONS The crystal structure of the composite Abl SH3-SH2 domain provides the first indication of how SH2 and SH3 domains communicate with each other within the same molecule and why the presence of one directly influences the activity of the other. This is the first clear evidence that these two domains are in contact with each other. The results suggest that this direct interaction between the two domains may affect the ligand binding properties of the SH2 domain, thus providing an explanation for biochemical and functional data concerning the Bcr-Abl kinase.

Mutational and structural analyses of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath)

BACKGROUND The soluble methane monooxygenase (sMMO) system in methanotrophic bacteria uses three protein components to catalyze the selective oxidation of methane to methanol. The coupling protein B (MMOB) both activates the carboxylate-bridged diiron center in the hydroxylase (MMOH) for substrate oxidation and couples the reaction to electron transfer from NADH through the sMMO reductase. Although the X-ray structure of the hydroxylase is known, little structural information is available regarding protein B. RESULTS Wild-type protein B from Methylococcus capsulatus (Bath) is very susceptible to degradation. The triple mutant protein B, Gly10-->Ala, Gly13-->Gln, Gly16-->Ala is resistant to degradation. Analyzing wild-type and mutant forms of protein B using size exclusion chromatography and circular dichroism spectroscopy suggests that the amino terminus of MMOB (Ser1-Ala25) is responsible for the proteolytic sensitivity and unusual mobility of the protein. We used the stable triple glycine protein B mutant to generate an affinity column for the hydroxylase and investigated the interaction between MMOH and MMOB. These results suggest the interaction is dominated by hydrophobic contacts. CONCLUSIONS A structural model is presented for protein B that explains both its proclivity for degradation and its anomalous behavior during size exclusion chromatography. The model is consistent with previously published biophysical data, including the NMR structure of the phenol hydroxylase regulatory protein P2. Furthermore, this model allows for detailed and testable predictions about the structure of protein B and the role of proposed recognition sites for the hydroxylase.

Crystallization and preliminary X-ray analysis of the methane monooxygenase hydroxylase protein from Methylococcus capsulatus (Bath)☆

Methane monooxygenase is a multicomponent enzyme system that catalyzes the conversion of methane to methanol in methanotrophic bacteria. Catalysis occurs at non-heme dinuclear iron centers contained in the hydroxylase component of the system, a dimer of composition alpha 2 beta 2 gamma 2. The hydroxylase protein from Methylococcus capsulatus (Bath) has been crystallized from aqueous solutions containing polyethylene glycol, lithium sulfate, and ammonium acetate. The crystals are orthorhombic, space group P2(1)2(1)2(1), with one dimer of relative molecular mass M(r) = 252,000 in the asymmetric unit. The unit cell dimensions are a = 62.6 A, b = 110.1 A, c = 333.5 A. The crystals diffract uniformly beyond 2.5 A resolution. Crystals of the related hydroxylase from Methylosinus trichosporium OB3b have also been obtained.

Carboxylate Shifts in the Active Site of the Hydroxylase Component of Soluble Methane Monooxygenase from Methylococcus capsulatus (Bath)

Carboxylate anions constitute an especially versatile group of ligands in inorganic chemistry. Numerous complexes in which the electron lone pairs of a carboxylate anion are coordinated to one or more metal ions have been reported (R. C. Mehrotra, R. Bohra, 1983).