A monomeric mutant of Clostridium symbiosum glutamate dehydrogenase: comparison with a structured monomeric intermediate obtained during refolding.

The refolding of Clostridium symbiosum glutamate dehydrogenase (GDH) involves the formation of an inactive structured monomeric intermediate prior to its concentration-dependent association. The structured monomer obtained after removal of guanidinium chloride was stable and competent for reconstitution into active hexamers. Site-directed mutagenesis of C. symbiosum gdh gene was performed to replace the residues Arg-61 and Phe-187 which are involved in subunit-subunit interactions, as determined by three-dimensional structure analysis. Heterologous over-expression in Escherichia coli of the double mutant (R61E/F187D) led to the production of a soluble protein with a molecular mass consistent with the monomeric form of clostridial GDH. This protein is catalytically inactive but cross-reacts with an anti-wild-type GDH antibody preparation. The double mutant R61E/F187D does not assemble into hexamers. The physical properties and the stability toward guanidinium chloride and urea of R61E/F187D were studied and compared to those of the structured monomeric intermediate.     

Three-dimensional structure of a monomeric form of a retroviral protease

The assembly of Mason-Pfizer monkey virus Gag polyproteins into immature capsids and their cleavage by the encoded protease are temporally and spatially separated processes, making the virus a particularly useful model for investigation of protease activation. Here we present a high resolution NMR structure of a fully folded monomer of a 12 kDa M-PMV protease (wt 12 PR) and of a Cys7Ala/Asp26Asn/Cys106Ala mutant (12 PR(D26N/C7A/C106A)). The overall structures of both wt 12 PR and 12 PR(D26N/C7A/C106A) follow the conservative structural motif of other retroviral proteases. The most prominent difference from the canonical fold of retroviral proteases is the absence of the interfacial beta-sheet, which leads to the loss of the principal force stabilizing the dimer of M-PMV PR. The monomer-dimer equilibrium can be shifted in favor of the dimer by adding a substrate or an inhibitor, partially compensating for the missing role of the beta-sheet. We also show that cysteines C7 and C106 play a crucial role in stabilizing the dimer and consequently increasing the proteolytic activity of M-PMV PR. This is consistent with the role of reversible oxidative modification of the cysteine residues in the regulation of the maturation of assembled M-PMV capsids in the cytoplasm.     

CD83 is a dimer: Comparative analysis of monomeric and dimeric isoforms

Recently, we reported that soluble CD83 has a strong immunosuppressive activity in vitro as well as in vivo. Sequence alignment of CD83 between different species revealed the presence of five cysteines in the extracellular Ig-domain of the protein. This opens up the possibility that four cysteines are involved in the formation of two intramolecular disulfide bonds and a possible involvement of the remaining fifth cysteine in the formation of an intermolecular covalent disulfide bond, leading to the dimerization of the extracellular protein domains. Using recombinant mutational analyses, where the fifth cytosine at amino acid position 129 was mutated to a serine, we could prove that the fifth cysteine residue was indeed necessary for the dimerization. Functional analyses revealed that the mutant protein inhibited almost completely the upregulation of CD83-expression during DC maturation. Furthermore, the functional activity of the mutant protein was investigated using MLR assays and we could show that the mutant soluble CD83 protein inhibited DC-mediated allogeneic T-cell stimulation in vitro.     

NMR structure of the unliganded Bombyx mori pheromone-binding protein at physiological pH

The nuclear magnetic resonance structure of the unliganded pheromone-binding protein (PBP) from Bombyx mori at pH above 6.5, BmPBP(B), consists of seven helices with residues 3-8, 16-22, 29-32, 46-59, 70-79, 84-100, and 107-124, and contains the three disulfide bridges 19-54, 50-108, and 97-117. This polypeptide fold encloses a large hydrophobic cavity, with a sufficient volume to accommodate the natural ligand bombykol. The polypeptide folds in free BmPBP(B) and in crystals of a BmPBP-bombykol complex are nearly identical, indicating that the B-form of BmPBP in solution represents the active conformation for ligand binding.     

Plasticity of quaternary structure: twenty-two ways to form a LacI dimer

The repressor proteins of the LacI/GalR family exhibit significant similarity in their secondary and tertiary structures despite less than 35% identity in their primary sequences. Furthermore, the core domains of these oligomeric repressors, which mediate dimerization, are homologous with the monomeric periplasmic binding proteins, extending the issue of plasticity to quaternary structure. To elucidate the determinants of assembly, a structure-based alignment has been created for three repressors and four periplasmic binding proteins. Contact maps have also been constructed for the three repressor interfaces to distinguish any conserved interactions. These analyses show few strict requirements for assembly of the core N-subdomain interface. The interfaces of repressor core C-subdomains are well conserved at the structural level, and their primary sequences differ significantly from the monomeric periplasmic binding proteins at positions equivalent to LacI 281 and 282. However, previous biochemical and phenotypic analyses indicate that LacI tolerates many mutations at 281. Mutations at LacI 282 were shown to abrogate assembly, but for Y282D this could be compensated by a second-site mutation in the core N-subdomain at K84 to L or A. Using the link between LacI assembly and function, we have further identified 22 second-site mutations that compensate the Y282D dimerization defect in vivo. The sites of these mutations fall into several structural regions, each of which may influence assembly by a different mechanism. Thus, the 360-amino acid scaffold of LacI allows plasticity of its quaternary structure. The periplasmic binding proteins may require only minimal changes to facilitate oligomerization similar to the repressor proteins.     

Structure of bovine rhodopsin in a trigonal crystal form

We have determined the structure of bovine rhodopsin at 2.65 A resolution using untwinned native crystals in the space group P3(1), by molecular replacement from the 2.8 A model (1F88) solved in space group P4(1). The new structure reveals mechanistically important details unresolved previously, which are considered in the membrane context by docking the structure into a cryo-electron microscopy map of 2D crystals. Kinks in the transmembrane helices facilitate inter-helical polar interactions. Ordered water molecules extend the hydrogen bonding networks, linking Trp265 in the retinal binding pocket to the NPxxY motif near the cytoplasmic boundary, and the Glu113 counterion for the protonated Schiff base to the extracellular surface. Glu113 forms a complex with a water molecule hydrogen bonded between its main chain and side-chain oxygen atoms. This can be expected to stabilise the salt-bridge with the protonated Schiff base linking the 11-cis-retinal to Lys296. The cytoplasmic ends of helices H5 and H6 have been extended by one turn. The G-protein interaction sites mapped to the cytoplasmic ends of H5 and H6 and a spiral extension of H5 are elevated above the bilayer. There is a surface cavity next to the conserved Glu134-Arg135 ion pair. The cytoplasmic loops have the highest temperature factors in the structure, indicative of their flexibility when not interacting with G protein or regulatory proteins. An ordered detergent molecule is seen wrapped around the kink in H6, stabilising the structure around the potential hinge in H6. These findings provide further explanation for the stability of the dark state structure. They support a mechanism for the activation, initiated by photo-isomerisation of the chromophore to its all-trans form, that involves pivoting movements of kinked helices, which, while maintaining hydrophobic contacts in the membrane interior, can be coupled to amplified translation of the helix ends near the membrane surfaces.     

Toward the solution structure of human insulin: sequential 2D 1H NMR assignment of a des-pentapeptide analogue and comparison with crystal structure

2D 1H NMR studies are presented of des-pentapeptide-insulin, an analogue of human insulin lacking the C-terminal five residues of the B chain. Removal of these residues, which are not required for function, is shown to reduce conformational broadening previously described in the spectrum of intact insulin [Weiss et al. (1989) Biochemistry 28, 9855-9873]. This difference presumably reflects more rapid internal motions in the fragment, which lead to more complete averaging of chemical shifts on the NMR time scale. Sequential 1H NMR assignment and preliminary structural analysis demonstrate retention in solution of the three alpha-helices observed in the crystal state and the relative orientation of the receptor-binding surfaces. These studies provide a foundation for determining the solution structure of insulin.     

A new B-chain mutant of insulin: comparison with the insulin crystal structure and role of sulfonate groups in the B-chain structure.

The solution structure of a new B-chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D-NMR and molecular modeling. This structure is compared with that of the oxidized B-chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res.46, 424-433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B-chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B-chain. 2D-NMR experiments confirmed, among multiple conformations, that the B-chain mutant presents defined secondary structures such as a alpha-helix between residues B9 and B19, and a beta-turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B-chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B-chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B-chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B-chains.     

The swapping of terminal arms in ribonucleases: comparison of the solution structure of monomeric bovine seminal and pancreatic ribonucleases

Bovine seminal ribonuclease (BS-RNase), the only dimeric protein among the pancreatic-like ribonucleases, is endowed with special structural features and with biological functions beyond enzymatic activity. In solution, the protein exists as an equilibrium mixture of two forms, with or without exchange (or swapping) of the N-terminal arms. After selective reduction and alkylation of the two intrachain disulfide bridges, the dimeric protein can be transformed into a monomeric derivative that has a ribonuclease activity higher than that of the parent dimeric protein but is devoid of the special biological functions. A detailed investigation of the structural features of this protein in solution, in comparison with those of other monomeric ribonucleases, may help unveil the structural details which induce swapping of the N-terminal arms of BS-RNase. The solution structure of the recombinant monomeric form of BS-RNase, as determined by 3D heteronuclear NMR, shows close similarity with that of bovine pancreatic ribonuclease (RNase A) in all regions characterized by regular elements of secondary structure. However, significant differences are present in the flexible regions, which could account for the different behavior of the two proteins. To characterize in detail these regions, we have measured H/D exchange rate constants, temperature coefficients and heteronuclear NOEs of backbone amides for both RNase A and monomeric BS-RNase. The results indicate a large difference in the backbone flexibility of the hinge peptide segment 16-22 of the two proteins, which could provide the molecular basis to explain the ability of BS-RNase subunits to swap their N-terminal arms.     

The periplasmic domain of TolR from Haemophilus influenzae forms a dimer with a large hydrophobic groove: NMR solution structure and comparison to SAXS data

TolR is a part of the Pal/Tol system which forms a five-member, membrane-spanning, multiprotein complex that is conserved in Gram-negative bacteria. The Pal/Tol system helps to maintain the integrity of the outer membrane and has been proposed to be involved in several other cellular processes including cell division. Obtaining the structure of TolR is of interest not only to help explain the many proposed functions of the Pal/Tol system but also to gain an understanding of the TolR homologues ExbD and MotB and to provide more targets for antibacterial treatments. In addition, the structure may provide insights into how colicins and bacteriophages are able to enter the cell. Here we report the solution structure of the homodimeric periplasmic domain of TolR from Haemophilus influenzae, determined with conventional, NOE-based NMR spectroscopy, supplemented by extensive residual dipolar coupling measurements. A novel method for assembling the dimer from small-angle X-ray scattering data confirms the NMR-derived structure. To facilitate NMR spectral analysis, a TolR construct containing residues 59-130 of the 139-residue protein was created. The periplasmic domain of TolR forms a C 2-symmetric dimer consisting of a strongly curved eight-stranded beta-sheet, generating a large deep groove on one side, while four helices cover the other face of the sheet. The structure of the TolR dimer together with data from the literature suggests how the periplasmic domain of TolR is most likely oriented relative to the cytoplasmic membrane and how it may interact with other components of the Pal/Tol system, particularly TolQ.     

Complex formation by bovine trypsin and a tetrapeptide (Leu-Arg-Pro-Gly-NH2): X-ray structure analysis of the complex in the orthorhombic crystal form with low molecular packing density

The title tetrapeptide, Leu-Arg-Pro-Gly-NH₂, forms a complex with trypsin in a novel orthorhombic crystal form with low molecular packing density. The complex formation was directly evidenced by X-ray crystallography. The crystal structure at 1.8 Å resolution was refined to anR-factor of 20.5% for 13,923 reflection data, which were measured with synchrotron radiation. The tetrapeptide is bound to trypsin at the active site, and the binding mode is very similar to that of a bovine pancreatic trypsin inhibitor (BPTI):trypsin complex. The tetrapeptide:trypsin complex is the first observation that a peptide forms a stable complex with trypsin.     

Properties and crystal structure of a beta-barrel folding mutant

A mutant of a beta-barrel protein, rat intestinal fatty acid binding protein, was predicted to be more stable than the wild-type protein due to a novel hydrogen bond. Equilibrium denaturation studies indicated the opposite: the V60N mutant protein was less stable. The folding transitions followed by CD and fluorescence were reversible and two-state for both mutant and wild-type protein. However, the rates of denaturation and renaturation of V60N were faster. During unfolding, the initial rate was associated with 75-80% of the fluorescence and all of the CD amplitude change. A subsequent rate accounted for the remaining fluorescence change for both proteins; thus the intermediate state lacked secondary structure. During folding, one rate was detected by both fluorescence and CD after an initial burst phase for both wild-type and mutant. An additional slower folding rate was detected by fluorescence for the mutant protein. The structure of the V60N mutant has been obtained and is nearly identical to prior crystal structures of IFABP. Analysis of mean differences in hydrogen bond and van der Waals interactions did not readily account for the stability loss due to the mutation. However, significant average differences of the solvent accessible surface and crystallographic displacement factors suggest entropic destabilization.     

Natural monomeric form of fetal bovine serum acetylcholinesterase lacks the C-terminal tetramerization domain

Acetylcholinesterase isolated from fetal bovine serum (FBS AChE) was previously characterized as a globular tetrameric form. Analysis of purified preparations of FBS AChE by gel permeation chromatography revealed the presence of a stable, catalytically active, monomeric form of this enzyme. The two forms could be distinguished from each other based on their molecular weight, hydrodynamic properties, kinetic properties, thermal stability, and the type of glycans they carry. No differences between the two forms were observed for the binding of classical inhibitors such as edrophonium and propidium or inhibitors that are current or potential drugs for the treatment of Alzheimer's disease such as (-) huperzine A and E2020; tacrine inhibited the monomeric form 2-3-fold more potently than the tetrameric form. Sequencing of peptides obtained from an in-gel tryptic digest of the monomer and tetramer by tandem mass spectrometry indicated that the tetramer consists of 583 amino acid residues corresponding to the mature form of the enzyme, whereas the monomer consists of 543-547 amino acid residues. The subunit molecular weight of the protein component of the monomer (major species) was determined to be 59 414 Da and that of the tetramer as 64 239 Da. The N-terminal of the monomer and the tetramer was Glu, suggesting that the monomer is not a result of truncation at the N-terminal. The only differences detected were at the C-terminus. The tetramer yielded the expected C-terminus, CSDL, whereas the C-terminus of the monomer yielded a mixture of peptides, of which LLSATDTLD was the most abundant. These results suggest that monomeric FBS AChE is trimmed at the C-terminus, and the results are consistent with the involvement of C-terminal amino acids in the assembly of monomers into tetramers.     

Solution 1H NMR of the active site of substrate-bound, cyanide-inhibited human heme oxygenase. comparison to the crystal structure of the water-ligated form

The majority of the active site residues of cyanide-inhibited, substrate-bound human heme oxygenase have been assigned on the basis of two-dimensional NMR using the crystal structure of the water-ligated substrate complex as a guide (Schuller, D. J., Wilks, A., Ortiz de Montellano, P. R., and Poulos, T. L. (1999) Nat. Struct. Biol. 6, 860-867). The proximal helix and the N-terminal portion of the distal helix are found to be identical to those in the crystal except that the heme for the major isomer ( approximately 75-80%) in solution is rotated 180 degrees about the alpha-gamma-meso axis relative to the unique orientation in the crystal. The central portion of the distal helix in solution is translated slightly over the heme toward the distal ligand, and a distal four-ring aromatic cluster has moved 1-2 A closer to the heme, which allows for strong hydrogen bonds between the hydroxyls of Tyr-58 and Tyr-137. These latter interactions are proposed to stabilize the closed pocket conducive to the high stereospecificity of the alpha-meso ring opening. The determination of the magnetic axes, for which the major axis is controlled by the Fe-CN orientation, reveals a approximately 20 degrees tilt of the distal ligand from the heme normal in the direction of the alpha-meso bridge, demonstrating that the close placement of the distal helix over the heme exerts control of stereospecificity by both blocking access to the beta, gamma, and delta-meso positions and tilting the axial ligand, a proposed peroxide, toward the alpha-meso position.     

Engineering the quaternary structure of an enzyme: construction and analysis of a monomeric form of malate dehydrogenase from Escherichia coli.

The citric acid cycle enzyme, malate dehydrogenase (MDH), is a dimer of identical subunits. In the crystal structures of 2 prokaryotic and 2 eukaryotic forms, the subunit interface is conformationally homologous. To determine whether or not the quaternary structure of MDH is linked to the catalytic activity, mutant forms of the enzyme from Escherichia coli have been constructed. Utilizing the high-resolution structure of E. coli MDH, the dimer interface was analyzed critically for side chains that were spatially constricted and needed for electrostatic interactions. Two such residues were found, D45 and S226. At their nearest point in the homodimer, they are in different subunits, hydrogen bond across the interface, and do not interact with any catalytic residues. Each residue was mutated to a tyrosine, which should disrupt the interface because of its large size. All mutants were cloned and purified to homogeneity from an mdh- E. coli strain (BHB111). Gel filtration of the mutants show that D45Y and D45Y/S226Y are both monomers, whereas the S226Y mutant remains a dimer. The monomeric D45Y and D45Y/S226Y mutants have 14,000- and 17,500-fold less specific activity, respectively, than the native enzyme. The dimeric S226Y has only 1.4-fold less specific activity. All forms crystallized, indicating they were not random coils. Data have been collected to 2.8 A resolution for the D45Y mutant. The mutant is not isomorphous with the native protein and work is underway to solve the structure by molecular replacement.     

Overexpression of a monomeric form of the bovine odorant-binding protein protects Escherichia coli from chemical-induced oxidative stress

Abstract

Mammalian odorant-binding proteins (OBPs) are soluble lipocalins produced in the nasal mucosa and in other epithelial tissues of several animal species, where they are supposed to serve as scavengers for small structurally unrelated hydrophobic molecules. These would include odorants and toxic aldehydes like 4-hydroxy-2-nonenal (HNE), which are end products of lipid peroxidation; therefore OBP might physiologically contribute to preserve the integrity of epithelial tissues under oxidative stress conditions by removing toxic compounds from the environment and, eventually, driving them to the appropriate degradative pathways. With the aim of developing a biological model based on a living organism for the investigation of the antioxidant properties of OBP, here we asked whether the overexpression of the protein could confer protection from chemical-induced oxidative stress in Escherichia coli. To this aim, bacteria were made to overexpress either GCC-bOBP, a redesigned monomeric mutant of bovine OBP, or its amino-terminal 6-histidine-tagged version 6H-GCC-bOBP. After inducing overexpression for 4 h, bacterial cells were diluted in fresh culture media, and their growth curves were followed in the presence of hydrogen peroxide (H₂O₂) and tert-Butyl hydroperoxide (tBuOOH), two reactive oxygen species whose toxicity is mainly due to lipid peroxidation, and menadione, a redox-cycling drug producing the superoxide ion. GCC-bOBP and 6H-GCC-bOBP were found to protect bacterial cells from the insulting agents H₂O₂ and tBuOOH but not from menadione. The obtained data led us to hypothesize that the presence of overexpressed OBP may contribute to protect bacterial cells against oxidative stress probably by sequestering toxic compounds locally produced during the first replication cycles by lipid peroxidation, before bacteria activate their appropriate enzyme-based antioxidative mechanisms.