Three-Dimensional Triple-Resonance NMR of \u3csup\u3e13\u3c/sup\u3eC/\u3csup\u3e15\u3c/sup\u3eN-Enriched Proteins Using Constant-Time Evolution

Recently it has been convincingly demonstrated that 30 triple-resonance NMR provides a practical alternative for obtaining sequential resonance assignments in larger proteins ( 1, 2). This approach requires a set of five or six 30 NMR experiments that correlate the various protein backbone nuclei. Details regarding the mechanisms and technical implementations of these experiments have been described previously ( 3- 5). Two of the experiments used in this approach correlate backbone Hα and Cα resonances with either the intraresidue carbonyl resonance (CO) or the 15N resonance of the succeeding residue and are referred to as HCACO and HCA(CO)N, respectively. The present Communication describes a modification of these experiments which optimizes their sensitivity and removes the F1 antiphase character of correlations

Relationships between the Precision of High-Resolution Protein NMR Structures, Solution-Order Parameters, and Crystallographic B Factors

One of the principal motivations for studying proteins by nuclear magnetic resonance stems from the desire to describe the solution structure of these molecules as compared to the generally perceived static picture obtained by X-ray crystallography. Indeed, it is one of the unique features of NMR spectroscopy that in addition to structural data, dynamic properties can be probed and characterized by measuring relaxation parameters. Furthermore, any mobility of the protein in solution will necessarily modulate the measured NMR parameters and should influence the resulting structure. It has been argued that regions of a protein that are highly mobile would be expected to be defined to a lesser degree of precision than regions that are rigid (1. 2 )

Molecular basis of human 46X,Y sex reversal revealed from the three-dimensional solution structure of the human SRY-DNA complex

AbstractThe solution structure of the specific complex between the high mobility group (HMG) domain of SRY (hSRY-HMG), the protein encoded by the human testis-determining, gene, and its DNA target site in the promoter of the Müllerian inhibitory substance gene has been determined by multidimensional NMR spectroscopy. hSRY-HMG has a twisted L shape that presents a concave surface (made up of three helices and the N- and C-terminal strands) to the DNA for sequence-specific recognition. Binding of hSRY-HMG to its specific target site occurs exclusively in the minor groove and induces a large conformational change in the DNA. The DNA in the complex has an overall 70°–80° bend and is helically unwound relative to classical A- and B-DNA. The structure of the complex reveals the origin of sequence-specific binding within the HMG-1/HMG-2 family and provides a framework for understanding the effects of point mutations that cause 46X,Y sex reversal at the atomic level

Point mutations of human interleukin-1 with decreased receptor binding affinity

AbstractInterleukin-1 (IL-1) is a monocyte-derived polypeptide hormone that interacts with a plasma membrane receptor. We have used oligonucleotide-directed mutagenesis to construct mutant human IL-1 proteins. Three different point mutants in a unique histidine residue (position 30) exhibited varying degrees of reduced IL-1 receptor binding affinity, whereas point mutants at five other residues behaved normally. Structural analysis of these mutant proteins by nuclear magnetic resonance spectroscopy detected no (or only minor) conformational changes relative to wild-type IL-1. These data suggest that the unique histidine residue influ- ences the architecture of the receptor binding site on human IL-1

Solution structure of human thioredoxin in a mixed disulfide intermediate complex with its target peptide from the transcription factor NFκB

AbstractBackground: Human thioredoxin is a 12 kDa cellular redox protein that plays a key role in maintaining the redox environment of the cell. It has recently been shown to be responsible for activating the DNA-binding properties of the cellular transcription factor, NFκB, by reducing a disulfide bond involving Cys62 of the p50 subunit. Using multidimensional heteronuclear-edited and heteronuclear-filtered NMR spectroscopy, we have solved the solution structure of a complex of human thioredoxin and a 13-residue peptide extending from residues 56–68 of p50, representing a kinetically stable mixed disulfide intermediate along the reaction pathway.Results The NFκB peptide is located in a long boot-shaped cleft on the surface of human thioredoxin delineated by the active-site loop, helices α2, α3 and α4, and strands β3 and β4. The peptide adopts a crescent-like conformation with a smooth 110° bend centered around residue 60 which permits it to follow the path of the cleft.Conclusion In addition to the intermolecular disulfide bridge between Cys32 of human thioredoxin and Cys62 of the peptide, the complex is stabilized by numerous hydrogen-bonding, electrostatic and hydrophobic interactions which involve residues 57–65 of the NFκB peptide and confer substrate specificity. These structural features permit one to suggest the specificity requirements for human thioredoxin-catalyzed disulfide bond reduction of proteins

The solution structure of human thioredoxin complexed with its target from Ref-1 reveals peptide chain reversal

AbstractBackground: Human thioredoxin (hTRX) is a 12 kDa cellular redox protein that has been shown to play an important role in the activation of a number of transcriptional and translational regulators via a thiol-redox mechanism. This activity may be direct or indirect via another redox protein known as Ref-1. The structure of a complex of hTRX with a peptide comprising its target from the transcription factor NFκB has previously been solved. To further extend our knowledge of the recognition by and interaction of hTRX with its various targets, we have studied a complex between hTRX and a Ref-1 peptide. This complex represents a kinetically stable mixed disulfide intermediate along the reaction pathway.Results Using multidimensional heteronuclear edited and filtered NMR spectroscopy, we have solved the solution structure of a complex between hTRX and a 13-residue peptide comprising residues 59–71 of Ref-1. The Ref-1 peptide is located in a crescent-shaped groove on the surface of hTRX, the groove being formed by residues in the active-site loop (residues 32–36), helix 3, β strands 3 and 5, and the loop between β strands 3 and 4. The complex is stabilized by numerous hydrogen-bonding and hydrophobic interactions that involve residues 61–69 of the peptide and confer substrate specificity.Conclusion The orientation of the Ref-1 peptide in the hTRX–Ref-1 complex is opposite to that found in the previously solved complex of hTRX with the target peptide from the transcription factor NFκB. Orientation is determined by three discriminating interactions involving the nature of the residues at the P−2, P−4 and P−5 binding positions. (P0 defines the active cysteine of the peptide, Cys65 for Ref-1 and Cys62 for NFκB. Positive and negative numbers indicate residues N-terminal and C-terminal to this residue, respectively, and vice versa for NFκB as it binds in the opposite orientation.) The environment surrounding the reactive Cys32 of hTRX, as well as the packing of the P+3 to P−4 residues are essentially the same in the two complexes, despite the opposing orientation of the peptide chains. This versatility in substrate recognition permits hTRX to act as a wide-ranging redox regulator for the cell

A detailed picture of a protein–carbohydrate hydrogen-bonding network revealed by NMR and MD simulations

Cyanovirin-N (CV-N) is a cyanobacterial lectin with antiviral activity towards HIV and several other viruses. Here, we identify mannoside hydroxyl protons that are hydrogen bonded to the protein backbone of the CV-N domain B binding site, using NMR spectroscopy. For the two carbohydrate ligands Manα(1→2)ManαOMe and Manα(1→2) Manα(1→6)ManαOMe five hydroxyl protons are involved in hydrogen-bonding networks. Comparison with previous crystallographic results revealed that four of these hydroxyl protons donate hydrogen bonds to protein backbone carbonyl oxygens in solution and in the crystal. Hydrogen bonds were not detected between the side chains of Glu41 and Arg76 with sugar hydroxyls, as previously proposed for CV-N binding of mannosides. Molecular dynamics simulations of the CV-N/Manα(1→2)Manα(1→6)ManαOMe complex confirmed the NMR-determined hydrogen-bonding network. Detailed characterization of CV-N/mannoside complexes provides a better understanding of lectin-carbohydrate interactions and opens up to the use of CV-N and similar lectins as antiviral agents

The Structure of the Cataract-Causing P23T Mutant of Human γD-Crystallin Exhibits Distinctive Local Conformational and Dynamic Changes†,‡

Crystallins are major proteins of the eye lens and essential for lens transparency. Mutations and aging of crystallins cause cataracts, the predominant cause of blindness in the world. In human γD-crystallin, the P23T mutant is associated with congenital cataracts. Until now, no atomic structural information has been available for this variant. Biophysical analyses of this mutant protein have revealed dramatically reduced solubility compared to that of the wild-type protein due to self-association into higher-molecular weight clusters and aggregates that retain a nativelike conformation within the monomers [Pande, A., et al. (2005) Biochemistry 44, 2491−2500]. To elucidate the structure and local conformation around the mutation site, we have determined the solution structure and characterized the protein’s dynamic behavior by NMR. Although the global structure is very similar to the X-ray structure of wild-type γD-crystallin, pivotal local conformational and dynamic differences are caused by the threonine substitution. In particular, in the P23T mutant, the imidazole ring of His22 switches from the predominant Nε2 tautomer in the wild-type protein to the Nδ1 tautomer, and an altered motional behavior of the associated region in the protein is observed. The data support structural changes that may initiate aggregation or polymerization by the mutant protein.National Institutes of Health (U.S.) (Grant GM 17980)National Eye Institute (Grant EY 015834