Resonance assignment and secondary structure determination and stability of the recombinant human uteroglobin with heteronuclear multidimensional NMR

Human uteroglobin (h-UG) or Clara cell 10kDa (cc10kDa) is a steroid-dependent, 17 kDahomodimeric, secretory protein with potent anti-inflammatory/immunomodulatory properties.However, the exact physiological role still remains to be determined. It has been hypothesisedthat its activity is exerted through the binding of a specific target represented by a smallmolecule (still unknown), and that the binding is regulated by the formation/disruption of twocysteine bonds. The binding properties of the reduced UG have been proved in vitro forseveral different molecules, but no in vivo data are available to date. However, binding hasbeen observed between reduced rabbit UG and a protein of an apparent molecular mass of90 kDa and, more recently, we found an h-UG-binding protein (putative receptor), of anapparent molecular mass of 190 kDa, on the surface of several cell types. The recognitioninvolves oxidised h-UG. These findings pose the problem of the relevance of the oxidationstate in the recognition process. To determine the solution structure of the oxidised h-UG, weproduced wild-type as well as uniformly 15N- and 15N/13C-labelled samples of therecombinant protein. The assignments of the 1H, 15N and 13C resonances are presented,based on a series of homonuclear 2D and 3D and heteronuclear 2D and 3D double and tripleresonance NMR experiments. Our results indicate that h-UG is an extremely stable proteinunder a wide range of temperatures and pH conditions. The secondary structure in solutionis in general agreement with previously reported crystal structures of rabbit UG, suggestingthat cc10kDa and h-UG are indeed the same protein. Small local differences found in the N-and C-terminal helices seem to support the hypothesis that flexibility involves these residues;moreover, it possibly accounts for the residual binding properties observed when the proteinis in the oxidised state.     

X-ray structure of human stromelysin catalytic domain complexed with nonpeptide inhibitors: implications for inhibitor selectivity.

Effective inhibitors of matrix metalloproteinases (MMPs), a family of connective tissue-degrading enzymes, could be useful for the treatment of diseases such as cancer, multiple sclerosis, and arthritis. Many of the known MMP inhibitors are derived from peptide substrates, with high potency in vitro but little selectivity among MMPs and poor bioavailability. We have discovered nonpeptidic MMP inhibitors with improved properties, and report here the crystal structures of human stromelysin-1 catalytic domain (SCD) complexed with four of these inhibitors. The structures were determined and refined at resolutions ranging from 1.64 to 2.0 A. Each inhibitor binds in the active site of SCD such that a bulky diphenyl piperidine moiety penetrates a deep, predominantly hydrophobic S'1 pocket. The active site structure of the SCD is similar in all four inhibitor complexes, but differs substantially from the peptide hydroxamate complex, which has a smaller side chain bound in the S'1 pocket. The largest differences occur in the loop forming the "top" of this pocket. The occupation of these nonpeptidic inhibitors in the S'1 pocket provides a structural basis to explain their selectivity among MMPs. An analysis of the unique binding mode predicts structural modifications to design improved MMP inhibitors.     

Solution secondary structure of calcium-saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy.

The solution secondary structure of calcium-saturated skeletal troponin C (TnC) in the presence of 15% (v/v) trifluoroethanol (TFE), which has been shown to exist predominantly as a monomer (Slupsky CM, Kay CM, Reinach FC, Smillie LB, Sykes BD, 1995, Biochemistry 34, forthcoming), has been investigated using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The 1H, 15N, and 13C NMR chemical shift values for TnC in the presence of TFE are very similar to values obtained for calcium-saturated NTnC (residues 1-90 of skeletal TnC), calmodulin, and synthetic peptide homodimers. Moreover, the secondary structure elements of TnC are virtually identical to those obtained for calcium-saturated NTnC, calmodulin, and the synthetic peptide homodimers, suggesting that 15% (v/v) TFE minimally perturbs the secondary and tertiary structure of this stably folded protein. Comparison of the solution structure of calcium-saturated TnC with the X-ray crystal structure of half-saturated TnC reveals differences in the phi/psi angles of residue Glu 41 and in the linker between the two domains. Glu 41 has irregular phi/psi angles in the crystal structure, producing a kink in the B helix, whereas in calcium-saturated TnC, Glu 41 has helical phi/psi angles, resulting in a straight B helix. The linker between the N and C domains of calcium-saturated TnC is flexible in the solution structure.     

Comparison of the NMR solution structure with the X-ray crystal structure of the activation domain from procarboxypeptidase B

Summary The NMR solution structure of the activation domain isolated from porcine procarboxypeptidase B is compared with the X-ray crystal structure of the corresponding segment in the intact proenzyme. For the region of the polypeptide chain that has a well-defined three-dimensional structure in solution, i.e., the backbone atoms of residues 11–76 and 25 amino acid side chains in this segment that form a hydrophobic core in the activation domain, the root-mean-square distance between the two structures is 1.1 Å. There are no significant differences in average atom positions between the two structures, but only the NMR structure shows increased structural disorder in three outlying loops located along the same edge of the activation domain. These regions of increased structural disorder in the free domain coincide only partially with the interface to the enzyme domain in the proenzyme.     

Crystal structures of the catalytic domain of human stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) with a hydroxamic acid inhibitor SM-25453

Crystal structures of the catalytic domain of human stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) with a hydroxamic acid inhibitor SM-25453 have been solved at 2.01 and 2.37A resolutions, respectively. The results revealed that the binding modes for this inhibitor to MMP-3 and -13 were quite similar. However, subtle comparative differences were observed at the bottom of S1' pockets, which were occupied with the guanidinomethyl moiety of the inhibitor. A remarkable feature of the inhibitor was the deep penetration of its long aliphatic chain into the S1' pocket and exposure of the guanidinomethyl moiety to the solvent.     

Proteolytic E-cadherin activation followed by solution NMR and X-ray crystallography

Cellular adhesion by classical cadherins depends critically on the exact proteolytic removal of their N-terminal prosequences. In this combined solution NMR and X-ray crystallographic study, the consequences of propeptide cleavage of an epithelial cadherin construct (domains 1 and 2) were followed at atomic level. At low protein concentration, the N-terminal processing induces docking of the tryptophan-2 side-chain into a binding pocket on the same molecule. At high concentration, cleavage induces dimerization (KD=0.72 mM, k(off)=0.7 s(-1)) and concomitant intermolecular exchange of the betaA-strands and the tryptophan-2 side-chains. Thus, the cleavage represents the switch from a nonadhesive to the functional form of cadherin.     

Structure of the Oct-3 POU-homeodomain in solution,as determined by triple resonance heteronuclear multidimensional NMR spectroscopy.

The POU-homeodomain (POUH) forms the bipartite DNA-binding POU domain in association with the POU-specific domain. The 1H, 15N, and 13C magnetic resonances of the 67-amino acid long POUH of mouse Oct-3 have almost completely been assigned, mainly through the combined use of three-dimensional triple resonance NMR methods. Based on the distance and dihedral angle constraints derived from the NMR data, the solution structure of the POUH domain has been calculated by the ab initio simulated annealing method. The average RMS deviation for all backbone heavy atoms of the 20 best calculated structures for residues 9-53 of the total 67 amino acid residues is 0.44 A. The POUH domain consists of three alpha-helices (helix-I, 10-20; helix-II, 28-38; and helix-III, 42-53), and helices-II and -III form a helix-turn-helix motif. In comparison with other classical homeodomains, the folding of the three helices is quite similar. However, the length of helix-III is fairly short. In the complex of the Oct-1 POU domain with an octamer site (Klemm JD, et al., 1994, Cell 77:21-32), the corresponding region is involved in helix-III. The structural difference between these two cases will be discussed.     

Effect of protein structural integrity on cross-linking by tyrosinase evidenced by multidimensional heteronuclear magnetic resonance spectroscopy

Enzymatic cross-linking of proteins can be catalyzed either by transferase-type enzymes, e.g., transglutaminases, or by oxidoreductases, e.g., tyrosinases or laccases. Three-dimensional structure of protein substrate plays a key role in these reactions, that is, the reactivity and end product are strongly modulated by the accessibility of target amino acid residues to the cross-linking enzyme. Typically structural integrity of protein can be distorted by heat, pH, or mechanical action, as well as by varying ionic concentration of the solution. In this study we used partially unfolded protein (wild-type DrkN SH3) and its structurally stabilized mutant (T22G) to investigate the impact of folded/unfolded conformations on cross-linking by Trichoderma reesei tyrosinase. Our results clearly showed formation of intermolecular cross-links solely between unfolded conformations, making them superior substrates to folded proteins when using tyrosinase as a cross-linking enzyme. Multidimensional heteronuclear magnetic resonance experiments in solution state were employed to investigate cross-linked end-products. The results presented in this study form basis for application development in food, medical, cosmetic, textile, packing and other sectors. In addition, the outcome of this study has a high value for the basic understanding of reaction mechanism of tyrosinases on proteins.     

1H and 15N NMR assignment and solution structure of the SH3 domain of spectrin: Comparison of unrefined and refined structure sets with the crystal structure

The assignment of the ¹H and ¹⁵Nnuclear magnetic resonance spectra of the Src-homology region 3 domain ofchicken brain -spectrin has been obtained. A set of solutionstructures has been determined from distance and dihedral angle restraints,which provide a reasonable representation of the protein structure insolution, as evaluated by a principal component analysis of the globalpairwise root-mean-square deviation (rmsd) in a large set of structuresconsisting of the refined and unrefined solution structures and the crystalstructure. The solution structure is well defined, with a lower degree ofconvergence between the structures in the loop regions than in the secondarystructure elements. The average pairwise rmsd between the 15 refinedsolution structures is 0.71 ± 0.13 Å for the backbone atoms and1.43 ± 0.14 Å for all heavy atoms. The solution structure isbasically the same as the crystal structure. The average rmsd between the 15refined solution structures and the crystal structure is 0.76 Å forthe backbone atoms and 1.45 ± 0.09 Å for all heavy atoms. Thereare, however, small differences probably caused by intermolecular contactsin the crystal structure.     

Structural insights into P-glycoprotein (ABCB1) by small angle X-ray scattering and electron crystallography

P-glycoprotein (ABCB1) is an ATP-binding cassette protein that is associated with the acquisition of multi-drug resistance in cancer and the failure of chemotherapy in humans. Structural insights into this protein are described using a combination of small angle X-ray scattering data and cryo-electron crystallography data. We have compared the structures with bacterial homologues, and discuss the development of homology models for P-glycoprotein based on the bacterial Sav1866 structure.     

综述: X射线晶体衍射是解析出青蒿素三维结构的唯一方法

X射线晶体衍射是解析出青蒿素三维结构的唯一方法     

Three-dimensional solution structure of mouse [Cd7]-metallothionein-1 by homonuclear and heteronuclear NMR spectroscopy

Sequential 1H-NMR assignments of mouse [Cd7]-metallothionein-1 (MT1) have been carried out by standard homonuclear NMR methods and the use of an accordion-heteronuclear multiple quantum correlation (HMQC) experiment for establishing the metal, 113Cd2+, to cysteine connectivities. The three-dimensional structure was then calculated using the distance constraints from two-dimensional nuclear Overhauser effect (NOE) spectroscopy spectra and the Cys-Cd connectivities as input for a distance geometry-dynamical simulated annealing protocol in X-PLOR 3.851. Similar to the mammalian MT2 isoforms, the homologous primary structure of MT1 suggested two separate domains, each containing one metal cluster. Because there were no interdomain constraints, the structure calculation for the N-terminal beta- and the C-terminal alpha-domain were carried out separately. The structures are based on 409 NMR constraints, consisting of 381 NOEs and 28 cysteine-metal connectivities. The only elements of regular secondary structure found were two short stretches of 3(10) helices along with some half-turns in the alpha-domain. Structural comparison with rat liver MT2 showed high similarity, with the beta-domain structure in mouse MT1 showing evidence of increased flexibility compared to the same domain in MT2. The latter was reflected by the presence of fewer interresidue NOEs, no slowly exchanging backbone amide protons, and enhanced cadmium-cadmium exchange rates found in the beta-domain of MT1.     

Comparison of the decameric structure of peroxiredoxin-II by transmission electron microscopy and X-ray crystallography.

The decameric human erythrocyte protein torin is identical to the thiol-specific antioxidant protein-II (TSA-II), also termed peroxiredoxin-II (Prx-II). Single particle analysis from electron micrographs of Prx-II molecules homogeneously orientated across holes in the presence of a thin film of ammonium molybdate and trehalose has facilitated the production of a >/=20 A 3-D reconstruction by angular reconstitution that emphasises the D5 symmetry of the ring-like decamer. The X-ray structure for Prx-II was fitted into the transmission electron microscopic reconstruction by molecular replacement. The surface-rendered transmission electron microscopy (TEM) reconstruction correlates well with the solvent-excluded surface of the X-ray structure of the Prx-II molecule. This provides confirmation that transmission electron microscopy of negatively stained specimens, despite limited resolution, has the potential to reveal a valid representation of surface features of protein molecules. 2-D crystallisation of the Prx-II protein on mica as part of a TEM study resulted in the formation of a p2 crystal form with parallel linear arrays of stacked rings. This latter 2-D form correlates well with that observed from the 2.7 A X-ray structure of Prx-II solved from a new orthorhombic 3-D crystal form.     

Bioactive conformation of a potent stromelysin inhibitor determined by X-nucleus filtered and multidimensional NMR spectroscopy

The biologically active conformation of a novel, very potent, nonpeptidic stromelysin inhibitor was determined by X-nucleus filtered and multidimensional NMR spectroscopy. This bound conformer was subsequently docked into the stromelysin catalytic domain (SCD) using intermolecular distance constraints derived from NOE data. The complex showed the S1′ pocket of stromelysin to be the major site of enzyme-inhibitor interaction with other portions of the inhibitor spanning the S2′ and S1 binding sites. Theoretical predictions of SCD-inhibitor binding from molecular modeling studies were consistent with the NMR data. Comparison of modeled enzyme-inhibitor complexes for stromelysin and collagenase revealed an alternate binding mode for the inhibitor in collagenase, suggesting a similar binding interaction might also be possible for stromelysin. The NMR results, however, revealed a single SCD-inhibitor binding mode and provided a structural template for the design of more potent stromelysin inhibitors.     

Crystallization and preliminary X-ray investigation of recombinant human interleukin 10

Crystals of recombinant human interleukin 10 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space group P4₁2₁2 or P4₃2₁2; the unit cell axes are a = 36.5 Å and c = 221.9 Å. There is the equivalent of one polypeptide chain in the asymmetric unit. The crystals are stable to X-rays and diffract to at least 2.5 Å resolution. © 1995 Wiley-Liss, Inc.     

Redesign of a four-helix bundle protein by phage display coupled with proteolysis and structural characterization by NMR and X-ray crystallography

To test whether it is practical to use phage display coupled with proteolysis for protein design, we used this approach to convert a partially unfolded four-helix bundle protein, apocytochrome b(562), to a stably folded four-helix bundle protein. Four residues expected to form a hydrophobic core were mutated. One residue was changed to Trp to provide a fluorescence probe for studying the protein's physical properties and to partially fill the void left by the heme. The other three positions were randomly mutated. In addition, another residue in the region to be redesigned was substituted with Arg to provide a specific cutting site for protease Arg-c. This library of mutants was displayed on the surface of phage and challenged with protease Arg-c to select stably folded proteins. The consensus sequence that emerged from the selection included hydrophobic residues at only one of the three positions and non-hydrophobic residues at the other two. Nevertheless, the selected proteins were thermodynamically very stable. The structure of a selected protein was characterized using multi-dimensional NMR. All four helices were formed in the structure. Further, site-directed mutagenesis was used to change one of the two non-hydrophobic residues to a hydrophobic residue, which increased the stability of the protein, indicating that the selection result was not based solely on the protein's global stability and that local structural characteristics may also govern the selection. This conclusion is supported by the crystal structure of another mutant that has two hydrophobic residues substituted for the two non-hydrophobic residues. These results suggest that the hydrophobic interactions in the core are not sufficient to dictate the selection and that the location of the cutting site of the protease also influences the selection of structures.     

NMR secondary structure and interactions of recombinant human MOZART1 protein,a component of the gamma‐tubulin complex

Mitotic‐spindle organizing protein associated with a ring of γ‐tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ‐tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC‐MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha‐helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N‐terminus (residues 1–250) of GCP3 (γ‐tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.     

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor.

Stromelysin, a representative matrix metalloproteinase and target of drug development efforts, plays a prominent role in the pathological proteolysis associated with arthritis and secondarily in that of cancer metastasis and invasion. To provide a structural template to aid the development of therapeutic inhibitors, we have determined a medium-resolution structure of a 20-kDa complex of human stromelysin's catalytic domain with a hydrophobic peptidic inhibitor using multinuclear, multidimensional NMR spectroscopy. This domain of this zinc hydrolase contains a mixed beta-sheet comprising one antiparallel strand and four parallel strands, three helices, and a methionine-containing turn near the catalytic center. The ensemble of 20 structures was calculated using, on average, 8 interresidue NOE restraints per residue for the 166-residue protein fragment complexed with a 4-residue substrate analogue. The mean RMS deviation (RMSD) to the average structure for backbone heavy atoms is 0.91 A and for all heavy atoms is 1.42 A. The structure has good stereochemical properties, including its backbone torsion angles. The beta-sheet and alpha-helices of the catalytic domains of human stromelysin (NMR model) and human fibroblast collagenase (X-ray crystallographic model of Lovejoy B et al., 1994b, Biochemistry 33:8207-8217) superimpose well, having a pairwise RMSD for backbone heavy atoms of 2.28 A when three loop segments are disregarded. The hydroxamate-substituted inhibitor binds across the hydrophobic active site of stromelysin in an extended conformation. The first hydrophobic side chain is deeply buried in the principal S'1 subsite, the second hydrophobic side chain is located on the opposite side of the inhibitor backbone in the hydrophobic S'2 surface subsite, and a third hydrophobic side chain (P'3) lies at the surface.