Backbone assignments and secondary structure of the Escherichia coli enzyme-II mannitol A domain determined by heteronuclear three-dimensional NMR spectroscopy.

This report presents the backbone assignments and the secondary structure determination of the A domain of the Escherichia coli mannitol transport protein, enzyme-IImtl. The backbone resonances were partially assigned using three-dimensional heteronuclear 1H NOE 1H-15N single-quantum coherence (15N NOESY-HSQC) spectroscopy and three-dimensional heteronuclear 1H total correlation 1H-15N single-quantum coherence (15N TOCSY-HSQC) spectroscopy on uniformly 15N enriched protein. Triple-resonance experiments on uniformly 15N/13C enriched protein were necessary to complete the backbone assignments, due to overlapping 1H and 15N frequencies. Data obtained from three-dimensional 1H-15N-13C alpha correlation experiments (HNCA and HN(CO)CA), a three-dimensional 1H-15N-13CO correlation experiment (HNCO), and a three-dimensional 1H alpha-13C alpha-13CO correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of virtually all observed backbone resonances. Determination of the secondary structure of IIAmtl is based upon NOE information from the 15N NOESY-HSQC and the 1H alpha and 13C alpha secondary chemical shifts. The resulting secondary structure is considerably different from that reported for IIAglc of E. coli and Bacillus subtilis determined by NMR and X-ray.     

Two-dimensional NMR studies of staphylococcal nuclease. 2. Sequence-specific assignments of carbon-13 and nitrogen-15 signals from the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

Samples of staphylococcal nuclease H124L (cloned protein overproduced in Escherichia coli whose sequence is identical with that of the nuclease isolated from the V8 strain of Staphylococcus aureus) were labeled uniformly with carbon-13 (26% ul 13C), uniformly with nitrogen-15 (95% ul 15N), and specifically by incorporating nitrogen-15-labeled leucine ([98% 15N]Leu) or carbon-13-labeled lysine ([26% ul 13C]Lys), arginine ([26% ul 13C]Arg), or methionine ([26% ul 13C]Met). Solutions of the ternary complexes of these analogues (nuclease H124L-pdTp-Ca2+) at pH 5.1 (H2O) or pH* 5.5 (2H2O) at 45 degrees C were analyzed as appropriate to the labeling pattern by multinuclear two-dimensional (2D) NMR experiments at spectrometer fields of 14.09 and 11.74 T: 1H-13C single-bond correlation (1H[13C]SBC); 1H-13C single-bond correlation with NOE relay (1H[13C]SBC-NOE); 1H-13C single-bond correlation with Hartmann-Hahn relay (1H-[13C]SBC-HH); 1H-13C multiple-bond correlation (1H[13C]MBC); 1H-15N single-bond correlation (1H-[15N]SBC); 1H-15N single-bond correlation with NOE relay (1H[15N]SBC-NOE). The results have assisted in spin system assignments and in identification of secondary structural elements. Nuclear Overhauser enhancements (NOE's) characteristic of antiparallel beta-sheet (d alpha alpha NOE's) were observed in the 1H [13C]-SBC-NOE spectrum of the nuclease ternary complex labeled uniformly with 13C. NOE's characteristic of alpha-helix (dNN NOE's) were observed in the 1H[15N]SBC-NOE spectrum of the complex prepared from protein labeled uniformly with 15N. The assignments obtained from these multinuclear NMR studies have confirmed and extended assignments based on 1H[1H] 2D NMR experiments [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry (preceding paper in this issue)].     

Structural dynamics of the membrane translocation domain of colicin E9 and its interaction with TolB

In order for the 61 kDa colicin E9 protein toxin to enter the cytoplasm of susceptible cells and kill them by hydrolysing their DNA, the colicin must interact with the outer membrane BtuB receptor and Tol translocation pathway of target cells. The translocation function is located in the N-terminal domain of the colicin molecule. (1)H, (1)H-(1)H-(15)N and (1)H-(13)C-(15)N NMR studies of intact colicin E9, its DNase domain, minimal receptor-binding domain and two N-terminal constructs containing the translocation domain showed that the region of the translocation domain that governs the interaction of colicin E9 with TolB is largely unstructured and highly flexible. Of the expected 80 backbone NH resonances of the first 83 residues of intact colicin E9, 61 were identified, with 43 of them being assigned specifically. The absence of secondary structure for these was shown through chemical shift analyses and the lack of long-range NOEs in (1)H-(1)H-(15)N NOESY spectra (tau(m)=200 ms). The enhanced flexibility of the region of the translocation domain containing the TolB box compared to the overall tumbling rate of the protein was identified from the relatively large values of backbone and tryptophan indole (15)N spin-spin relaxation times, and from the negative (1)H-(15)N NOEs of the backbone NH resonances. Variable flexibility of the N-terminal region was revealed by the (15)N T(1)/T(2) ratios, which showed that the C-terminal end of the TolB box and the region immediately following it was motionally constrained compared to other parts of the N terminus. This, together with the observation of inter-residue NOEs involving Ile54, indicated that there was some structural ordering, resulting most probably from the interactions of side-chains. Conformational heterogeneity of parts of the translocation domain was evident from a multiplicity of signals for some of the residues. Im9 binding to colicin E9 had no effect on the chemical shifts or other NMR characteristics of the region of colicin E9 containing the TolB recognition sequence, though the interaction of TolB with intact colicin E9 bound to Im9 did affect resonances from this region. The flexibility of the translocation domain of colicin E9 may be connected with its need to recognise protein partners that assist it in crossing the outer membrane and in the translocation event itself.     

Dynamical structure of the antibody combining site as studied by 1H-15N shift correlation NMR spectroscopy.

The Fv fragment, which is a smallest antigen-binding unit of immunoglobulin, has been used for a 1H-15N shift correlation NMR study of the dynamical structure of the antibody combining site. Fv has been prepared by clostripain digestion of a mouse anti-dansyl IgG2a monoclonal antibody that lacks the entire CH1 domain. We have previously reported that of the six hypervariable regions, three each from the heavy chain (H1, H2, and H3) and the light chain (L1, L2, and L3), H3 is primarily responsible for the antigen binding in the anti-dansyl Fv fragment. The backbone amide nitrogens of all non-proline amino acid residues in H3 have been multiply labeled with 15N. [15N]T2 relaxation times and hydrogen-deuterium exchange rates of the amide groups of the main chain were measured in the absence and presence of epsilon-dansyl-L-lysine (DNS-Lys). It has been shown that (1) in the absence of DNS-Lys H3 displays a significant degree of internal motion and (2) antigen binding induces a significant change in the dynamical structure of H3.     

Assignments of backbone 1H, 13C, and 15N resonances and secondary structure of ribonuclease H from Escherichia coli by heteronuclear three-dimensional NMR spectroscopy.

The assignments of individual magnetic resonances of backbone nuclei of a larger protein, ribonuclease H from Escherichia coli, which consists of 155 amino acid residues and has a molecular mass of 17.6 kDa are presented. To remove the problem of degenerate chemical shifts, which is inevitable in proteins of this size, three-dimensional NMR was applied. The strategy for the sequential assignment was, first, resonance peaks of amides were classified into 15 amino acid types by 1H-15N HMQC experiments with samples in which specific amino acids were labeled with 15N. Second, the amide 1H-15N peaks were connected along the amino acid sequence by tracing intraresidue and sequential NOE cross peaks. In order to obtain unambiguous NOE connectivities, four types of heteronuclear 3D NMR techniques, 1H-15N-1H 3D NOESY-HMQC, 1H-15N-1H 3D TOCSY-HMQC, 13C-1H-1H 3D HMQC-NOESY, and 13C-1H-1H 3D HMQC-TOCSY, were applied to proteins uniformly labeled either with 15N or with 13C. This method gave a systematic way to assign backbone nuclei (N, NH, C alpha H, and C alpha) of larger proteins. Results of the sequential assignments and identification of secondary structure elements that were revealed by NOE cross peaks among backbone protons are reported.     

Native and non-native secondary structure and dynamics in the pH 4 intermediate of apomyoglobin

The partly folded state of apomyoglobin at pH 4 represents an excellent model for an obligatory kinetic folding intermediate. The structure and dynamics of this intermediate state have been extensively examined using NMR spectroscopy. Secondary chemical shifts, (1)H-(1)H NOEs, and amide proton temperature coefficients have been used to probe residual structure in the intermediate state, and NMR relaxation parameters T(1) and T(2) and ?(1)H?-(15)N NOE have been analyzed using spectral densities to correlate motion of the polypeptide chain with these structural observations. A significant amount of helical structure remains in the pH 4 state, indicated by the secondary chemical shifts of the (13)C(alpha), (13)CO, (1)H(alpha), and (13)C(beta) nuclei, and the boundaries of this helical structure are confirmed by the locations of (1)H-(1)H NOEs. Hydrogen bonding in the structured regions is predominantly native-like according to the amide proton chemical shifts and their temperature dependence. The locations of the A, G, and H helix segments and the C-terminal part of the B helix are similar to those in native apomyoglobin, consistent with the early, complete protection of the amides of residues in these helices in quench-flow experiments. These results confirm the similarity of the equilibrium form of apoMb at pH 4 and the kinetic intermediate observed at short times in the quench-flow experiment. Flexibility in this structured core is severely curtailed compared with the remainder of the protein, as indicated by the analysis of the NMR relaxation parameters. Regions with relatively high values of J(0) and low values of J(750) correspond well with the A, B, G, and H helices, an indication that nanosecond time scale backbone fluctuations in these regions of the sequence are restricted. Other parts of the protein show much greater flexibility and much reduced secondary chemical shifts. Nevertheless, several regions show evidence of the beginnings of helical structure, including stretches encompassing the C helix-CD loop, the boundary of the D and E helices, and the C-terminal half of the E helix. These regions are clearly not well-structured in the pH 4 state, unlike the A, B, G, and H helices, which form a native-like structured core. However, the proximity of this structured core most likely influences the region between the B and F helices, inducing at least transient helical structure.     

Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence-specific assignments of hydrogen-1 signals and solution structure of the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

Staphylococcal nuclease H124L is a recombinant protein produced in Escherichia coli whose sequence is identical with that of the nuclease produced by the V8 variant of Staphylococcus aureus. The enzyme-metal ion activator-nucleotide inhibitor ternary complex, nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+, was investigated by two-dimensional (2D) NMR techniques. Efficient overproduction of the enzyme facilitated the production of random fractionally deuterated protein, which proved essential for detailed NMR analysis. 1H NMR spin systems were analyzed by conventional 2D 1H[1H] methods: COSY, relayed COSY, HOHAHA, and NOESY. Assignments obtained by 1H NMR experiments were confirmed and extended by 1H-13C and 1H-15N heteronuclear NMR experiments [Wang, J., Hinck, A. P., Loh, S. N., & Markley, J. L. (1990) Biochemistry (following paper in this issue)]. Spectra of the ternary complexes prepared with protein at natural abundance and at 50% random fractional deuteration provided the information needed for sequence-specific assignments of 121 of the 149 amino acid residues. Short- and intermediate-range NOE connectivities allowed the determination of secondary structural features of the ternary complex: three alpha-helical domains and three antiparallel beta-pleated sheets with several reverse turns. A number of nonsequential long-range HN-HN and H alpha-HN connectivities revealed additional information about the spatial arrangement of these secondary structural elements. The solution structure of this ternary complex shows a close correspondence to the crystal structure of the nuclease wt-thymidine 3',5'-bisphosphate-Ca2+ ternary complex [Cotton, F. A., Hazen, E. E., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555].     

Characterization of the structure and dynamics of amyloidogenic variants of human lysozyme by NMR spectroscopy

The structures and dynamics of the native states of two mutational variants of human lysozyme, I56T and D67H, both associated with non-neuropathic systemic amyloidosis, have been investigated by NMR spectroscopy. The (1)H and (15)N main-chain amide chemical shifts of the I56T variant are very similar to those of the wild-type protein, but those of the D67H variant are greatly altered for 28 residues in the beta-domain. This finding is consistent with the X-ray crystallographic analysis, which shows that the structure of this variant is significantly altered from that of the wild-type protein in this region. The (1)H-(15)N heteronuclear NOE values show that, with the exception of V121, every residue in the wild-type and I56T proteins is located in tightly packed structures characteristic of the native states of most proteins. In contrast, D67H has a region of substantially increased mobility as shown by a dramatic decrease in heteronuclear NOE values of residues near the site of mutation. Despite this unusual flexibility, the D67H variant has no greater propensity to form amyloid fibrils in vivo or in vitro than has I56T. This finding indicates that it is the increased ability of the variants to access partially folded conformations, rather than intrinsic changes in their native state properties, that is the origin of their amyloidogenicity.     

Disruption of an intermonomer salt bridge in the p53 tetramerization domain results in an increased propensity to form amyloid fibrils

We describe in molecular detail how disruption of an intermonomer salt bridge (Arg337-Asp352) leads to partial destabilization of the p53 tetramerization domain and a dramatically increased propensity to form amyloid fibrils. At pH 4.0 and 37 degrees C, a p53 tetramerization domain mutant (p53tet-R337H), associated with adrenocortical carcinoma in children, readily formed amyloid fibrils, while the wild-type (p53tet-wt) did not. We characterized these proteins by equilibrium denaturation, 13C(alpha) secondary chemical shifts, (1H)-15N heteronuclear NOEs, and H/D exchange. Although p53tet-R337H was thermodynamically less stable, NMR data indicated that the two proteins had similar secondary structure and molecular dynamics. NMR derived pK(a) values indicated that at low pH the R337H mutation partially disrupted an intermonomer salt bridge. Backbone H/D exchange results showed that for at least a small population of p53tet-R337H molecules disruption of this salt bridge resulted in partial destabilization of the protein. It is proposed that this decrease in p53tet-R337H stability resulted in an increased propensity to form amyloid fibrils.     

Assignment of the backbone 1H and 15N NMR resonances of bacteriophage T4 lysozyme

The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.     

Investigation of the backbone dynamics of the IgG-binding domain of streptococcal protein G by heteronuclear two-dimensional 1H-15N nuclear magnetic resonance spectroscopy.

The backbone dynamics of the immunoglobulin-binding domain (B1) of streptococcal protein G, uniformly labeled with 15N, have been investigated by two-dimensional inverse detected heteronuclear 1H-15N NMR spectroscopy at 500 and 600 MHz. 15N T1, T2, and nuclear Overhauser enhancement data were obtained for all 55 backbone NH vectors of the B1 domain at both field strengths. The overall correlation time obtained from an analysis of the T1/T2 ratios was 3.3 ns at 26 degrees C. Overall, the B1 domain is a relatively rigid protein, consistent with the fact that over 95% of the residues participate in secondary structure, comprising a four-stranded sheet arranged in a -1, +3x, -1 topology, on top of which lies a single helix. Residues in the turns and loops connecting the elements of secondary structure tend to exhibit a higher degree of mobility on the picosecond time scale, as manifested by lower values of the overall order parameter. A number of residues at the ends of the secondary structure elements display two distinct internal motions that are faster than the overall rotational correlation time: one is fast (< 20 ps) and lies in the extreme narrowing limit, whereas the other is one to two orders of magnitude slower (1-3 ns) and lies outside the extreme narrowing limit. The slower motion can be explained by large-amplitude (20-40 degrees) jumps in the N-H vectors between states with well-defined orientations that are stabilized by hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

15N NMR assignments and chemical shift analysis of uniformly labeled 15N calbindin D9k in the apo, (Cd2+)1 and (Ca2+)2 states.

15N has been uniformly incorporated into the EF-hand Ca(2+)-binding protein calbindin D9k so that heteronuclear experiments can be used to further characterize the structure and dynamics of the apo, (Cd2+)1 and (Ca2+)2 states of the protein. The 15N NMR resonances were assigned by 2D 15N-resolved 1H experiments, which also allowed the identification of a number of sequential and medium-range 1H-1H contacts that are obscured by chemical shift degeneracy in homonuclear experiments. The 15N chemical shifts are analyzed with respect to correlations with protein secondary structure. In addition, the changes in 15N chemical shift found for the apo----(Cd2+)1----(Ca2+)2 binding sequence confirm that the effects on the protein are mainly associated with chelation of the first ion.     

Multinuclear NMR study of the structure of the Fv fragment of anti-dansyl mouse IgG2a antibody

A multinuclear NMR study is reported of Fv, which is a minimum antigen-binding unit of immunoglobulin. Fv has been prepared by clostripain digestion of a mouse anti-dansyl IgG2a monoclonal antibody that lacks the entire CH1 domain [Takahashi, H., Igarashi, T., Shimada, I., & Arata, Y. (1991) Biochemistry 30, 2840-2847]. A variety of Fv analogues labeled with 2H in the aromatic rings and with 13C and/or 15N in the peptide bonds have been prepared and used for multinuclear NMR analyses of Fv in the absence and presence of epsilon-dansyl-L-lysine (DNS-Lys). It has been shown that 1H-15N shift correlation spectra of Fv sensitively reflect the antigen binding and can be used along with 1H and 13C spectral data for the structural analyses of antigen-antibody interactions. Hydrogen-deuterium exchange of the amide protons has been followed in the absence and presence of DNS-Lys by using the 1H-15N shift correlation spectra. Use of the beta-shift observed for the carbonyl carbon resonances has also been helpful in following the hydrogen-deuterium exchange. On the basis of the NMR data obtained, the static and dynamic structure of the Fv fragment in the absence and presence of DNS-Lys has been discussed.     

Assignment of the aliphatic 1H and 13C resonances of the Bacillus subtilis glucose permease IIA domain using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy.

Nearly complete assignment of the aliphatic 1H and 13C resonances of the IIAglc domain of Bacillus subtilis has been achieved using a combination of double- and triple-resonance three-dimensional (3D) NMR experiments. A constant-time 3D triple-resonance HCA(CO)N experiment, which correlates the 1H alpha and 13C alpha chemical shifts of one residue with the amide 15N chemical shift of the following residue, was used to obtain sequence-specific assignments of the 13C alpha resonances. The 1H alpha and amide 15N chemical shifts had been sequentially assigned previously using principally 3D 1H-15N NOESY-HMQC and TOCSY-HMQC experiments [Fairbrother, W. J., Cavanagh, J., Dyson, H. J., Palmer, A. G., III, Sutrina, S. L., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1991) Biochemistry 30, 6896-6907]. The side-chain spin systems were identified using 3D HCCH-COSY and HCCH-TOCSY spectra and were assigned sequentially on the basis of their 1H alpha and 13C alpha chemical shifts. The 3D HCCH and HCA(CO)N experiments rely on large heteronuclear one-bond J couplings for coherence transfers and therefore offer a considerable advantage over conventional 1H-1H correlation experiments that rely on 1H-1H 3J couplings, which, for proteins the size of IIAglc (17.4 kDa), may be significantly smaller than the 1H line widths. The assignments reported herein are essential for the determination of the high-resolution solution structure of the IIAglc domain of B. subtilis using 3D and 4D heteronuclear edited NOESY experiments; these assignments have been used to analyze 3D 1H-15N NOESY-HMQC and 1H-13C NOESY-HSQC spectra and calculate a low-resolution structure [Fairbrother, W. J., Gippert, G. P., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1992) FEBS Lett. 296, 148-152].     

The secondary structure of the colicin E3 immunity protein as studied by 1H-1H and 1H-15N two-dimensional NMR spectroscopy.

By performing 1H-1H and 1H-15N two-dimensional (2D) nuclear magnetic resonance (NMR) experiments, the complete sequence-specific resonance assignment was determined for the colicin E3 immunity protein (84 residues; ImmE3), which binds to colicin E3 and inhibits its RNase activity. First, the fingerprint region of the spectrum was analyzed by homonuclear 1H-1H HOHAHA and NOESY methods. For the identification of overlapping resonances, heteronuclear 1H-15N (HMQC-HOHAHA, HMQC-NOESY) experiments were performed, so that the complete 1H and 15N resonance assignments were provided. Then the secondary structure of ImmE3 was determined by examination of characteristic patterns of sequential backbone proton NOEs in combination with measurement of exchange rates of amide protons and 3JHN alpha coupling constants. From these results, it was concluded that ImmE3 contains a four-stranded antiparallel beta-sheet (residues 2-10, 19-22, 47-49, and 71-79) and a short alpha-helix (residues 31-36).     

The NMR structure of the nucleocapsid protein from the mouse mammary tumor virus reveals unusual folding of the C-terminal zinc knuckle

The nucleocapsid protein (NC) from the mouse mammary tumor virus (MMTV) has been overexpressed in Escherichia coli and purified to homogeneity for structural studies by nuclear magnetic resonance (NMR) spectroscopy. The protein contains two copies of a conserved zinc-coordinating "CCHC array" or "zinc knuckle" motif common to the nucleocapsid proteins of nearly all known retroviruses. The residues comprising and adjacent to the zinc knuckles were assigned by standard two-dimensional (1)H and three-dimensional (1)H-(15)N NMR methods; the rotational dynamic properties of the protein were determined from (15)N relaxation experiments, and distance restraints derived from the nuclear Overhauser effect (NOE) data were used to calculate the three-dimensional structure. The (1)H-(1)H NOE and (15)N relaxation data indicate that the two zinc knuckles do not interact with each other, but instead behave as independently folded domains connected by a flexible 13-residue linker segment. The proximal zinc knuckle folds in a manner that is essentially identical to that observed previously for the two zinc knuckles of the human immunodeficiency virus type 1 nucleocapsid protein and for the moloney murine leukemia virus nucleocapsid zinc knuckle domain. However, the distal zinc knuckle of MMTV NC exhibits a rare three-dimensional fold that includes an additional C-terminal beta-hairpin. A similar C-terminal reverse turn-like structure was observed recently in the distal zinc knuckle of the Mason-Pfizer monkey virus nucleocapsid protein [Gao, Y., et al. (1998) Protein Sci. 7, 2265-2280]. However, despite a high degree of sequence homology, the conformation and orientation of the beta-hairpin in MMTV NC is significantly different from that of the reverse turn in MPMV NC. The results support the conclusion that structural features of NC zinc knuckle domains can vary significantly among the different genera of retroviridae, and are discussed in terms of the recent and surprising discovery that MMTV NC can facilitate packaging of the HIV-1 genome in chimeric MMTV mutants.     

Disease-causing mutations in cartilage oligomeric matrix protein cause an unstructured Ca2+ binding domain.

Chondrocytes from pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) patients display an enlarged rough endoplasmic reticulum that accumulates extracellular matrix proteins, including cartilage oligomeric matrix protein (COMP). Mutations that cause PSACH and EDM1 are restricted to a 27-kDa Ca(2+) binding domain (type 3 repeat). This domain has 13 Ca(2+)-binding loops with a consensus sequence that conforms to Ca(2+)-binding loops found in EF hands. Most disease-causing mutations are found in the 11-kDa C-terminal region of this domain. We expressed recombinant native and mutant forms of the type 3 repeat domain (T3) and its 11-kDa C-terminal region (T3-Cterm). T3 and T3-Cterm bind approximately 13 and 8 mol of Ca(2+)/mol of protein, respectively. CD, one-dimensional proton, and two-dimensional (1)H-(15)N HSQC spectra of Ca(2+)-bound T3-Cterm indicate a distinct conformation that has little helical secondary structure, despite the presence of 13 EF hand Ca(2+)-binding loops. This conformation is also formed within the context of the intact T3. 19 cross-peaks found between 9.0 and 11.4 ppm are consistent with the presence of strong hydrogen bonding patterns, such as those in beta-sheets. Removal of Ca(2+) leads to an apparent loss of structure as evidenced by decreased dispersion and loss of all down field resonances. Deletion of Asp-470 (a mutation found in 22% of all PSACH and EDM1 patients) decreased the Ca(2+)-binding capacity of both T3 and T3-Cterm by about 3 mol of Ca(2+)/mol of protein. Two-dimensional (1)H-(15)N HSQC spectra of mutated T3-Cterm showed little evidence of defined structure in the presence or absence of Ca(2+). The data demonstrate that Ca(2+) is required to nucleate folding and to maintain defined structure. Mutation results in a partial loss of Ca(2+)-binding capacity and prevents Ca(2+)-dependent folding. Persistence of an unstructured state of the mutated Ca(2+) binding domain in COMP is the structural basis for retention of COMP in the rough endoplasmic reticulum of differentiated PSACH and EDM1 chondrocytes.     

Guanine nucleotides differentially modulate backbone dynamics of the STAS domain of the SulP/SLC26 transport protein Rv1739c of Mycobacterium tuberculosis

Enzymatic catalysis and protein signaling are dynamic processes that involve local and/or global conformational changes occurring across a broad range of time scales. (1) H-(15) N relaxation NMR provides a comprehensive understanding of protein backbone dynamics both in the apo (unliganded) and ligand-bound conformations, enabling both fast and slow internal motions of individual amino acid residues to be observed. We recently reported the structure and nucleotide binding properties of the sulfate transporter and anti-sigma factor antagonist (STAS) domain of Rv1739c, a SulP anion transporter protein of Mycobacterium tuberculosis. In the present study, we report (1) H-(15) N NMR backbone dynamics measurements [longitudinal (T(1) ), transverse (T(2) ) and steady-state ({(1) H}-(15) N) heteronuclear NOE] of the Rv1739c STAS domain, in the absence and presence of saturating concentrations of GTP and GDP. Analysis of measured relaxation data and estimated dynamic parameters indicated distinct features differentiating the binding of GTP and GDP to Rv1739c STAS. The 9.55 ns overall rotational correlation time of Rv1739c STAS increased to 10.48 ns in the presence of GTP, and to 13.25 ns in the presence of GDP, indicating significant nucleotide-induced conformational changes. These conformational changes were accompanied by slow time scale (μs to ms) motions in discrete regions of the protein, as reflected by guanine nucleotide-induced changes in relaxation parameters. The observed nucleotide-specific alterations in the relaxation properties of individual STAS residues reflect an increased molecular anisotropy and/or the emergence of conformational equilibria governing functional properties of the STAS domain.     

Multinuclear magnetic resonance studies of Escherichia coli adenylate kinase in free and bound forms. Resonance assignment, secondary structure and ligand binding.

The crystal structure of Escherichia coli adenylate kinase (AKe) revealed three main components: a CORE domain, composed of a five-stranded parallel beta-sheet surrounded by alpha-helices, and two peripheral domains involved in covering the ATP in the active site (LID) and binding of the AMP (NMPbind). We initiated a long-term NMR study aiming to characterize the solution structure, binding mechanism and internal dynamics of the various domains. Using single (15N) and double-labeled (13C and 15N) samples and double- and triple-resonance NMR experiments we assigned 97% of the 1H, 13C and 15N backbone resonances, and proton and 13Cbeta resonances for more than 40% of the side chains in the free protein. Analysis of a 15N-labeled enzyme in complex with the bi-substrate analogue [P1,P5-bis(5'-adenosine)-pentaphosphate] (Ap5A) resulted in the assignment of 90% of the backbone 1H and 15N resonances and 42% of the side chain resonances. Based on short-range NOEs and 1H and 13C secondary chemical shifts, we identified the elements of secondary structure and the topology of the beta-strands in the unliganded form. The alpha-helices and the beta-strands of the parallel beta-sheet in solution have the same limits (+/- 1 residue) as those observed in the crystal. The first helix (alpha1) appears to have a frayed N-terminal side. Significant differences relative to the crystal were noticed in the LID domain, which in solution exhibits four antiparallel beta-strands. The secondary structure of the nucleoside-bound form, as deduced from intramolecular NOEs and the 1Halpha chemical shifts, is similar to that of the free enzyme. The largest chemical shift differences allowed us to map the regions of protein-ligand contacts. 1H/2H exchange experiments performed on free and Ap5A-bound enzymes showed a general decrease of the structural flexibility in the complex which is accompanied by a local increased flexibility on the N-side of the parallel beta-sheet.     

1H and 15N nuclear magnetic resonance assignment and secondary structure of the cytotoxic ribonuclease alpha-Sarcin.

The ribosome-inactivating protein alpha-Sarcin (alpha S) is a 150-residue fungal ribonuclease that, after entering sensitive cells, selectively cleaves a single phosphodiester bond in an universally conserved sequence of the major rRNA to inactivate the ribosome and thus exert its cytotoxic action. As a first step toward establishing the structure-dynamics-function relationships in this system, we have carried out the assignment of the 1H and 15N NMR spectrum of alpha S on the basis of homonuclear (1H-1H) and heteronuclear (1H-15N) two-dimensional correlation spectra of a uniformly 15N-labeled sample, and two selectively 15N-labeled (Tyr and Phe) samples, as well as a single three-dimensional experiment. The secondary structure of alpha S, as derived from the characteristic patterns of dipolar connectivities between backbone protons, conformational chemical shifts, and the protection of backbone amide protons against exchange, consists of a long N-terminal beta-hairpin, a short alpha-helical segment, and a C-terminal beta-sheet of five short strands arranged in a + 1, + 1, + 1, + 1 topology, connected by long loops in which the 13 Pro residues are located.