1H, 13C, 15N nuclear magnetic resonance backbone assignments and secondary structure of human calcineurin B

The calmodulin- and calcium-stimulated protein phosphatase calcineurin, PP2B, consists of two subunits: calcineurin B, which binds Ca2+, and calcineurin A, which contains the catalytic site and a calmodulin binding site. Heteronuclear 3D and 4D NMR experiments were carried out on a recombinant human calcineurin B which is a 170-residue protein of molecular mass 19.3 kDa, uniformly labeled with 15N and 13C. The nondenaturing detergent CHAPS was used to obtain a monomeric form of calcineurin B. Three-dimensional triple resonance experiments yielded complete sequential assignment of the backbone nuclei (1H, 13C, and 15N). This assignment was verified by a 4D HN(COCA)NH experiment carried out with 50% randomly deuteriated and uniformly 15N- and 13C-enriched calcineurin B. The secondary structure of calcineurin B has been determined on the basis of the 13C alpha and 13C beta secondary chemical shifts, J(HNH alpha) couplings, and NOE connectivities obtained from 3D 15N-separated and 4D 13C/15N-separated NOESY spectra. Calcineurin B has eight helices distributed in four EF-hand, helix-loop-helix [Kretsinger, R. H. (1980) CRC Crit. Rev. Biochem. 8, 119-174] calcium binding domains. The secondary structure of calcineurin B is highly homologous to that of calmodulin. In comparison to calmodulin, helices B and C are shorter while helix G is considerably longer. As was observed for calmodulin in solution, calcineurin B does not have a single long central helix; rather, helices D and E are separated by a six-residue sequence in a flexible nonhelical conformation.     

1H and 15N nuclear magnetic resonance assignments, secondary structure in solution, and solvent exchange properties of azurin from Alcaligenes denitrificans

Complete sequential 1H and 15N resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) = 14,000, 129 residues) from Alcaligenes denitrificans have been obtained at pH 5.5 and 32 degrees C using homo- and heteronuclear two-dimensional and heteronuclear three-dimensional NMR spectroscopy. Comparison of the resonance assignments for the backbone protons with those of Pseudomonas aeruginosa azurin, which is 68% homologous in its amino acid sequence and has a very similar three-dimensional structure, showed a high similarity in chemical shift positions. After adjustment for random coil contributions the mean difference in NH chemical shifts is 0.00 ppm (root mean square width = 0.30 ppm), whereas for C alpha protons the mean difference is 0.09 ppm (root mean square width = 0.23 ppm). Characteristic NOE connectivities and 3JHN alpha values were used to determine the secondary structure of azurin in solution. Two beta-sheets, one helix, and nine tight and four helical turns were identified, and some long-range NOE contacts were found that connect the helix with the beta-sheets. The secondary structure obtained is in agreement with the structure derived from X-ray diffraction data [Baker, E. N. (1988) J. Mol. Biol. 203, 1071-1095]. Studies of the hydration of the protein in the vicinity of the copper ligand residue His117 revealed that the solvent-exposed N epsilon 2 of His117 is in slow exchange with the bulk solvent. However, no evidence was obtained for the presence of a long-lived water molecule at the position corresponding to a well-defined water molecule observed in the crystal structures of A. denitrificans and Ps. aeruginosa azurin.     

Unusual helix-containing greek keys in development-specific Ca(2+)-binding protein S. 1H, 15N, and 13C assignments and secondary structure determined with the use of multidimensional double and triple resonance heteronuclear NMR spectroscopy

Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone and side-chain 1H, 15N, and 13C resonance assignments of calcium loaded Myxococcus xanthus protein S (173 residues). Of the range of constant-time triple resonance experiments recorded, HNCACB and CBCA(CO)NH, which correlate C alpha and C beta with backbone amide resonances of the same and the succeeding residue respectively, proved particularly useful in resolving assignment ambiguities created by the 4-fold internal homology of the protein S amino acid sequence. Extensive side-chain 1H and 13C assignments have been obtained by analysis of HCCH-TOCSY and 15N-edited TOCSY-HMQC spectra. A combination of NOE, backbone amide proton exchange, 3JNH alpha coupling constant, and chemical shift data has been used to show that each of the protein S repeat units consists of four beta-strands in a Greek key arrangement. Two of the Greek keys contain a regular alpha-helix between the third and fourth strands, resulting in an unusual and possibly unique variation on this common folding motif. Despite similarity between two nine-residue stretches in the first and third domains of protein S and one of the Ca(2+)-binding sequences in bovine brain calmodulin [Inouye, S., Franceschini, T., & Inouye, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6829-6833], the protein S topology in these regions is incompatible with an EF-hand calmodulin-type Ca(2+)-binding site.     

Sequential assignment of 1H, 15N, 13C resonances and secondary structure of human calmodulin-like protein determined by NMR spectroscopy

Human calmodulin-like protein (CLP) is closely related to vertebrate calmodulin, yet its unique cell specific expression pattern, overlapping but divergent biochemical properties, and specific target proteins suggest that it is not an isoform of calmodulin. To gain insight into the structural differences that may underlie the difference target specificities and biochemical properties of CLP when compared to calmodulin, we determined the sequential backbone assignment and associated secondary structure of 144 out of the 148 residues of Ca2+-CLP by using multinuclear multidimensional NMR spectroscopy. Despite a very high overall degree of structural similarity between CLP and calmodulin, a number of significant differences were found mainly in the length of alpha-helices and in the central nonhelical flexible region. Interestingly, the regions of greatest primary sequence divergence between CLP and calmodulin in helices III and VIII displayed only minor secondary structure differences. The data suggest that the distinct differences in target specificity and biochemical properties of CLP and calmodulin result from the sum of several minor structural and side-chain changes spread over multiple domains in these proteins.     

Proton nuclear magnetic resonance sequential assignments and secondary structure of an immunoglobulin light chain-binding domain of protein L

The 1H NMR assignments have been made for the immunoglobulin (Ig) light chain-binding B1 domain of protein L from Peptostreptococcus magnus. The secondary structure elements and the global folding pattern were determined from nuclear Overhauser effects, backbone coupling constants, and slowly exchanging amide protons. The B1 domain was found to be folded into a globular unit of 61 amino acid residues, preceded by a 15 amino acid long disordered N-terminus. The folded portion of the molecule contains a four-stranded beta-sheet spanned by a central alpha-helix. The fold is similar to the IgG-binding domains of streptococcal protein G, despite the fact that the binding sites on immunoglobulins for the two proteins are different; protein G binds IgG through the constant (Fc) part of the heavy chain, whereas protein L has affinity for the variable domain of Ig light chains.     

Localization of cellular retinoic acid-binding protein (CRABP) II and CRABP in developing rat testis

Retinoic acid (RA) has been implicated as a signaling molecule for the morphogenesis of some tissues and organs. The morphogenesis of the rat testis occurs relatively late in development, culminating in puberty. Two members of the superfamily of small intracellular carrier proteins for lipophilic compounds are cellular Ra-binding protein (CRABP) and cellular RA-binding protein II (CRABP-II). Both CRABP and CRABP-II are present at various sites in the developing mouse embryo. Here we report the developmental expression and localization of CRABP and CRABP-II in rat testis. Northern blot analysis of CRABP-II demonstrated the highest messenger RNA expression on day 4 (the earliest time point assayed by this technique), decreasing thereafter until day 20, when it became undetectable. Western blot analysis, begun on day 19 of fetal development, indicated that high levels of protein expression in the testis already existed at that time. CRABP messenger RNA expression reached its highest levels between postnatal days 16-20 and decreased thereafter. Immunolocalization revealed that CRABP-II was confined to the fetal population of Leydig and Sertoli cells. We observed that CRABP-II was expressed in certain cells that synthesized retinoic acid in the uterus and ovary (unpublished). The expression of CRABP-II in Sertoli cells and fetal Leydig cells suggested that these cells may well be the site of RA synthesis in the developing testis. CRABP was localized to gonocytes in earlier stages and spermatogonia later, where it was clearly excluded from the nucleus, indicating that the role of CRABP may be to protect these cells from the effects of RA. The reported expression of CRABP-II in embryonal tissues, which are RA responsive and undergoing morphogenesis, coupled with CRABP-II expression in the testis at a critical morphogenic stage suggest that RA may play a prominent role in the morphogenesis of the testis.     

1H and 15N resonance assignment of neural cell adhesion molecule module-2

This article presents a simple, inexpensive method for precisely locating the floor of the maxillary sinus, as well as the presence of any septa, at the time of sinus augmentation surgery. Using an anesthesia light wand placed transnasally to illuminate the sinus, the surgeon can reliably elevate the lateral maxillary wall overlying the sinus with relative ease without fear of placing the osteotomy cuts too far from the sinus floor. The same procedure can be used postoperatively to evaluate the density of the bone graft placed into the sinus prior to closure.     

The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates

The crystal structure of the recombinant form of rat liver fatty acid-binding protein was completed to 2.3 A and refined to an R factor of 19.0%. The structural solution was obtained by molecular replacement using superimposed polyalanine coordinates of six intracellular lipid-binding proteins as a search probe. The entire amino acid sequence of rat liver fatty acid-binding protein along with an amino-terminal formyl-methionine was modeled in the crystal structure. In addition, the crystal was obtained in the presence of oleic acid, and the initial electron density clearly showed two fatty acid molecules bound within a central cavity. The carboxylate of one fatty acid molecule interacts with arginine 122 and is shielded from free solvent. It has an overall bent conformation. The more solvent-exposed carboxylate of the other oleate is located near the helix-turn-helix that caps one end of the beta-barrel, while the acyl chain lies in the interior. The cavity contains both polar and nonpolar residues but also shows extensive hydrophobic character around the nonpolar atoms of the ligands. The primary and secondary oleate binding sites appear to be totally interdependent, mainly because favorable hydrophobic interactions form between both aliphatic chains.     

Complete heteronuclear NMR resonance assignments and secondary structure of core binding factor beta (1-141)

We report a patient who developed significant liver dysfunction following therapy with terbinafine. At the end of a 3 1/2-wk course of terbinafine, he developed progressive jaundice and pruritus. His serum bilirubin peaked at 30.9 mg/dl 3 wk after discontinuing terbinafine. A liver biopsy revealed mild to moderate mixed cellular infiltrate in the portal tracts, and hepatocellular and canicular cholestasis. His liver tests normalized 100 days after stopping terbinafine.     

Characterization of the three-dimensional solution structure of human profilin: 1H, 13C, and 15N NMR assignments and global folding pattern

Human profilin is a 15-kDa protein that plays a major role in the signaling pathway leading to cytoskeletal rearrangement. Essentially complete assignment of the 1H, 13C, and 15N resonances of human profilin have been made by analysis of multidimensional, double- and triple-resonance nuclear magnetic resonance (NMR) experiments. The deviation of the 13C alpha and 13C beta chemical shifts from their respective random coil values were analyzed and correlate well with the secondary structure determined from the NMR data. Twenty structures of human profilin were refined in the program X-PLOR using a total of 1186 experimentally derived conformational restraints. The structures converged to a root mean squared distance deviation of 1.5 A for the backbone atoms. The resultant conformational ensemble indicates that human profilin is an alpha/beta protein comprised of a seven-stranded, antiparallel beta-sheet and three helices. The secondary structure elements for human profilin are quite similar to those found in Acanthamoeba profilin I [Archer, S. J., Vinson, V. K., Pollard, T. D., & Torchia, D. A. (1993), Biochemistry 32, 6680-6687], suggesting that the three-dimensional structure of Acanthamoeba profilin I should be analogous to that determined here for human profilin. The structure determination of human profilin has facilitated the sequence alignment of lower eukaryotic and human profilins and provides a framework upon which the various functionalities of profilin can be explored. At least one element of the actin-binding region of human profilin is an alpha-helix. Two mechanisms by which phosphatidylinositol 4,5-bisphosphate can interfere with actin-binding by human profilin are proposed.     

1H-15N NMR signal assignment and secondary structure of bacteriorhodopsin (1-231) in solution

15N-Labeled de-(232-248)-bacteriorhodopsin [BR(1-231)] was solubilized in 1:1 chloroform-methanol solvent mixture that contained 1.0 M 2HCO2N2H4 and mimic membrane medium. Resonances in the 1H-15N heteronuclear multiple-quantum coherence (HMQC) spectrum of BR (1-231) were assigned using the data of two- and three-dimensional NMR experiments. Of 117 cross-peaks present in the 1H-15N HMQC spectrum, 98 were assigned to residues in 1-75 and 193-231 segments of the protein. Almost all cross-peaks that correspond to the 76-192 segment were absent in the HMQC spectrum (except for six cross-peaks from the side chains and 14 cross-peaks from the backbone). Deuterium exchange rates of amide protons and cross-peaks of nuclear Overhauser effect helped to localize helices A (residues 8-30), B (residues 40-65), and G (residues 198-226). The periodicity in the rates of deuterium exchange of NH protons of helices A, B, and G was explained by the compact arrangement of these helices in the protein globule. The broadening of signals from six residues in helix G, which, according to the electron cryomicroscopy model of bacteriorhodopsin, is in contact with the NMR-unobservable bundle of helices CDEF, indicates specific interactions of the helices in BR(1-231). These data suggest that BR(1-231) solubilized in an organic medium has a spatial structure similar to that in the electron cryomicroscopy model of BR.     

1H-NMR resonance assignments, secondary structure, and global fold of the TR1C fragment of turkey skeletal troponin C in the calcium-free state

The TR1C fragment of turkey skeletal muscle TnC (residues 12-87) comprises the two regulatory calcium binding sites of the protein. Complete assignments of the 1H-NMR resonances of the backbone and amino acid side chains of this domain in the absence of metal ions have been obtained using 2D 1H-NMR techniques. Sequential (i,i+1) and short-range (i,i+3) NOE connectivities define two helix-loop-helix calcium binding motifs, and long-range NOE connectivities indicate a short two-stranded beta-sheet formed between the two calcium binding loops. The two calcium binding sites are different in secondary structure. In terms of helix length, site II conforms to a standard "EF-hand" motif with the first helix ending one residue before the first calcium ligand and the second helix starting one residue after the beta-sheet. In site I, the first helix ends three residues before the first calcium ligand, and the second helix starts three residues after the beta-sheet. A number of long-range NOE connectivities between the helices define their relative orientation and indicate formation of a hydrophobic core between helices A, B, and D. The secondary structure and global fold of the TR1C fragment in solution in the calcium-free state are therefore very similar to those of the corresponding region in the crystal structure of turkey skeletal TnC [Herzberg, O., & James, M.N.G. (1988) J. Mol. Biol. 203, 761-779].     

Two-dimensional 1H and 15N NMR titration studies of hisactophilin

We have used two-dimensional 1H-15N heteronuclear single quantum correlation spectroscopy to measure the pH dependence of backbone amide group chemical shifts in the actin binding protein hisactophilin over the pH range 5.7-11.1. Most of the resonances can be analyzed using a simple equation involving a single apparent ionization constant, pK(app). The majority of resonances in the protein titrate with pK(app) values of 5.6-7.4. The results can be rationalized in terms of titration of many histidine residues in hisactophilin. The titration data provide direct experimental support for the proposed models of the atomic basis of actin and membrane binding by hisactophilin.