SMN oligomerization defect correlates with spinal muscular atrophy severity.

Spinal muscular atrophy (SMA) is a motor-neuron disorder resulting from anterior-horn-cell death. The autosomal recessive form has a carrier frequency of 1 in 50 and is the most common genetic cause of infant death. SMA is categorized as types I-III, ranging from severe to mild, based upon age of onset and clinical course. Two closely flanking copies of the survival motor neuron (SMN) gene are on chromosome 5q13 (ref. 1). The telomeric SMN (SMN1) copy is homozygously deleted or converted in >95% of SMA patients, while a small number of SMA disease alleles contain missense mutations within the carboxy terminus. We have identified a modular oligomerization domain within exon 6 of SMN1. All previously identified missense mutations map within or immediately adjacent to this domain. Comparison of wild-type to mutant SMN proteins of type I, II and III SMA patients showed a direct correlation between oligomerization and clinical type. Moreover, the most abundant centromeric SMN product, which encodes exons 1-6 but not 7, demonstrated reduced self-association. These findings identify decreased SMN self-association as a biochemical defect in SMA, and imply that disease severity is proportional to the intracellular concentration of oligomerization-competent SMN proteins.

Solution conformation of purine-pyrimidine DNA octamers using nuclear magnetic resonance, restrained molecular dynamics and NOE-based refinement.

The solution structures of two alternating purine-pyrimidine octamers, [d(G-T-A-C-G-T-A-C)]2 and the reverse sequence [d(C-A-T-G-C-A-T-G)]2, are investigated by using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Chemical shift assignments are obtained for non-exchangeable protons by a combination of two-dimensional correlation and nuclear Overhauser enhancement (NOE) spectroscopy experiments. Distances between protons are estimated by extrapolating distances derived from time-dependent NOE measurements to zero mixing time. Approximate dihedral angles are determined within the deoxyribose ring from coupling constants observed in one and two-dimensional spectra. Sets of distance and dihedral determinations for each of the duplexes form the bases for structure determination. Molecular dynamics is then used to generate structures that satisfy the experimental restraints incorporated as effective potentials into the total energy. Separate runs start from classical A and B-form DNA and converge to essentially identical structures. To circumvent the problems of spin diffusion and differential motion associated with distance measurements within molecules, models are improved by NOE-based refinement in which observed NOE intensities are compared to those calculated using a full matrix analysis procedure. The refined structures generally have the global features of B-type DNA. Some, but not all, variations in dihedral angles and in the spatial relationships of adjacent base-pairs are observed to be in synchrony with the alternating purine-pyrimidine sequence.

Role of the microcin B17 propeptide in substrate recognition: solution structure and mutational analysis of McbA1-26.

BACKGROUND: The peptide antibiotic microcin B17 (MccB17) contains oxazole and thiazole heterocycles formed by the post-translational modification of four cysteine and four serine residues. An amino-terminal propeptide targets the 69 amino acid precursor of MccB17 (preproMccB17) to the heterocyclization enzyme MccB17 synthetase. The mode of synthetase recognition has been unclear, because there has been limited structural information available on the MccB17 propeptide to date. RESULTS: The solution structure of the MccB17 propeptide (McbA1-26), determined using nuclear magnetic resonance, reveals that McbA1-26 is an amphipathic alpha helix. Mutational analysis of 13 propeptide residues showed that Phe8 and Leu12 are essential residues for MccB17 synthetase recognition. A domain of the propeptide was putatively identified as the region that interacts with the synthetase. CONCLUSIONS: MccB17 synthetase recognizes key hydrophobic residues within a helical propeptide, allowing the selective heterocyclization of downstream cysteine and serine residues in preproMccB17. The determination of the solution structure of the propeptide should facilitate the investigation of other functions of the propeptide, including a potential role in antibiotic secretion.

Effects of DNA binding and metal substitution on the dynamics of the GAL4 DNA-binding domain as studied by amide proton exchange.

Backbone amide proton exchange rates in the DNA-binding domain of GAL4 have been determined using 1H-15N heteronuclear correlation NMR spectroscopy. Three forms of the protein were studied-the native Zn-containing protein, the Cd-substituted protein, and a Zn-GAL4/DNA complex. Exchange rates in the Zn-containing protein are significantly slower than in the Cd-substituted protein. This shows that Cd-substituted GAL4 is destabilized relative to the native Zn-containing protein. Upon DNA binding, global retardation of amide proton exchange with solvent was observed, indicating that internal fluctuations of the DNA-recognition module are significantly reduced by the presence of DNA. In all forms of the protein, the internal dyad symmetry of the DNA-recognition module of GAL4 is reflected by the backbone amide proton exchange rates.

Characterization of gamma-radiation induced decomposition products of thymidine-containing dinucleoside monophosphates by nuclear magnetic resonance spectroscopy.

To study the chemical and biochemical influence of loss of base aromaticity, dinucleoside monophosphates containing cis-5R,6S-thymidine glycol (Tg) and 5R and 5S 5,6-dihydrothymidine (Th) were prepared from d-ApT and d-TpA by KMnO4 oxidation and rhodium-catalysed hydrogenation, respectively. One and two dimensional 1H NMR techniques were used to characterize the solution conformation of each of the modified dinucleoside monophosphates for comparison with the unmodified compounds. Coupling constant data show that all sugar moieties adopt a predominantly 2'-endo conformation. Estimates of proton-proton distances from two-dimensional NOE experiments reveal that most of the glycosidic bonds prefer the anti conformation. Analysis of the C5'-C4' (gamma) torsion angle of the hydroxymethyl group using 3JH4'H5' and 3JH4'H5" data indicate that these modifications to thymine have little effect on the gamma conformer populations. Although, in general, additions at C5 and C6 of thymine in d-ApT and d-TpA profoundly distort the pyrimidine, they do not otherwise significantly alter the conformation of these compounds relative to the unsubstituted dinucleoside monophosphates. The one exception is the thymine glycol of d-TgpA, which appears to have a higher syn population than the parent compound.

Structure determination of membrane-associated proteins from nuclear magnetic resonance data.

This Review covers the delineation and optimization of protein-lipid systems for study using solution-state NMR spectroscopy. The first half presents the necessary background for a membrane protein biochemist to initiate collaboration with an NMR spectroscopist. The second half provides guidelines for the spectroscopist on data collection, analysis for obtaining conformational information, and structure generation and assessment. Although the emphasis is on the study of peptides in detergent micelles, methods are outlined for larger membrane-associated proteins and for use of other solubilizing agents.

Membrane-binding peptide from the C2 domain of factor VIII forms an amphipathic structure as determined by NMR spectroscopy.

Factor VIII binds to cell membranes prior to assembling with the serine protease, factor IXa, to form the factor X-activating enzyme complex. In order to better understand the interaction between factor VIII and phosphatidylserine-containing membranes, we have synthesized the membrane-binding peptide from the C2 domain of factor VIII, corresponding to residues 2303-2324. The peptide, fVIII2303-24, with a primary structure of TRYLRIHPQSWVHQIALRMEVL, aggregates at concentrations above 2 microM at pH 7 but is soluble at pH 6. fVIII2303-24 competes with fluorescein-labeled factor VIII (Ki = 3 microM) for binding sites on synthetic phosphatidylserine-containing membranes and for binding sites on stimulated platelets. Circular dichroism spectra indicate that fVIII2303-24 is predominantly a random coil in aqueous solution but adopts a predominantly helical conformation upon interaction with SDS micelles. 1H NMR spectroscopy in the presence of SDS micelles allowed estimation of interproton distances from the nuclear Overhauser effect and estimation of torsion angles from coupling constants indicated by splitting of resonance lines. The distance and angle estimates, processed by distance geometry/simulated annealing software, indicate that fVIII2303-24 has an alpha-helical segment encompassing residues P8-E20 and an extended segment encompassing residues L4-P8. The location of six hydrophobic residues on one face of the structure suggests that hydrophobic interactions contribute to membrane-binding. In addition, two arginines penetrate the hydrophobic plane suggesting that they interact with phosphate moieties in a phospholipid bilayer.

A nuclear magnetic resonance study of the DNA-binding affinity of Cro repressor protein stabilized by a disulfide bond.

The structure, dynamics, and DNA-binding characteristics of wild-type and cross-linked Cro repressors are compared by using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The Cro repressor is a small dimeric DNA-binding protein from bacteriophage lambda. Replacement of valine-55 by cysteine in the dimer interaction region of each monomer subunit results in the spontaneous formation of a disulfide cross-link between the subunits. Two-dimensional nuclear Overhauser effect spectroscopy and CD data show the variant has nearly the same conformation as the wild-type protein. However, by monitoring the CD band at 222 nm, the cross-linked protein is shown to have a heat-denaturation midpoint temperature of 67 degrees C, whereas the wild-type protein has a melting temperature of about 47 degrees C. Using 1H-NMR to follow the denaturation by heat, the same melting temperature is observed for wild-type Cro (47 degrees C), but a much lower melting temperature is seen for V55C Cro (58 degrees C). This suggests that between 58 and 67 degrees C the cross-linked protein exists in a molten globule state with the alpha-helices mainly intact, but without the interaction of chemical groups that cause spectral dispersion. Binding parameters for interaction of the proteins with DNA were obtained by observing the NMR spectrum for the imino protons of a 10 base-pair half-operator DNA and titrating in protein. The cross-linked protein binds DNA (Kd = 160 microM) about eight times more weakly than the wild-type protein (Kd = 19 microM). Adjustments in protein structure, necessary to form a tight protein-DNA complex, appear to be hindered by a loss in protein flexibility caused by the intersubunit cross-link.

Comparison of the structures of operator DNA free and in complex with lambda repressor.

The structures of operator DNA unbound and in complex with lambda repressor protein are compared. The conformation of the left 10 base pairs of a lambda right regulatory operator DNA sequence has been previously determined in solution using nuclear magnetic resonance techniques and the structure of a homologous left regulatory operator DNA bound to lambda repressor N-terminal domain had been previously solved using X-ray crystallography. The DNA adopts an overall linear B-form DNA both in the absence and presence of lambda repressor. Superimpositioning of the DNA structures reveals small differences between them that are due to the binding of protein and not to the different techniques used for their determination.

A novel method for selective isotope labeling of bacterially expressed proteins.

A novel method for isotope labeling in selected amino acids is presented for use with the T7 RNA polymerase system. The protocol is illustrated with the DNA-binding domain from the E2 protein of bovine papillomavirus, BPV-1. On addition of rifampicin, protein expression occurs exclusively from the gene controlled by the T7 promoter. Since the bacteria are now dedicated to the production of E2 protein, labeling with specific amino acids is efficiently performed. For example, 10 mg/l of 15N-labeled phenylalanine is shown to be sufficient for incorporation of the label, without scrambling, and without the use of an auxotrophic strain.

Different interactions of Cro repressor dimer with the left and right halves of OR3 operator DNA.

lambda Cro repressor protein is titrated with two half-operator DNA duplexes comprising the right and left halves of the major binding site on phage lambda DNA, the OR3 operator. The comparison of binding strengths and the conformation of Cro repressor in the two protein-DNA complexes shows that base pair differences between the two halves of the OR3 operator affect the binding of Cro repressor protein. Some 1H NMR resonances are assigned for both protein and DNA in the Cro-operator DNA complexes which are then used to highlight differences between Cro right half and Cro left half protein-operator DNA interactions. These differences are compared to the asymmetry found in the lambda C1 repressor-operator DNA complex. Mechanisms for the recognition of the Cro transcriptional regulatory protein have considered only interactions between a single Cro monomer and a consensus half-operator site with the assumption that the interactions in the remaining half-site are related by the 2-fold symmetry of the complex. A revised model is suggested that allows asymmetry in the two halves of the protein-DNA complex. Methods are proposed to avoid problems in the general use of 1H NMR spectroscopy to study protein-DNA interaction such as intermediate exchange behavior and sample aggregation.

Refined solution structure of the DNA-binding domain of GAL4 and use of 3J(113Cd,1H) in structure determination.

We have refined the solution structure of cadmium-bound GAL4 and present its 15N and 1H NMR assignments. The root-mean-square (rms) deviation to the average structure was 0.4 +/- 0.05 A for backbone atoms, and 0.9 +/- 0.1 A for all heavy atoms. The three-bond heteronuclear 3J(113Cd,1H) coupling constants were found to disobey a Karplus-type relationship, which was attributable to the unusual constraints imposed by the bimetal-thiolate cluster in GAL4. We conclude that the structural parameters that correlate to 3J(113Cd,1H) are complex.

Structure and topography of the membrane-binding C2 domain of factor VIII in the presence of dodecylphosphocholine micelles.

A 21 residue peptide from the C2 domain of the antihaemophilic factor VIII competes with factor VIII for membrane-binding sites in vitro. Here, we provide the structure and topography of the peptide in solution, on dodecylphosphocholine (DPC) micelles, determined using 1H-NMR spectroscopy. The peptide assumes an amphipathic structure comprising an extended N-terminal region and a C-terminal helix. The average root-mean-square deviation is 0.7+/-0.2 A for the superimposition of the backbone atoms of Ile6 to Arg18 on the lowest energy structure. Whereas the backbone conformation is similar to that in SDS micelles, the Trp11 side-chain orientation is dramatically changed. The indole ring is nearly parallel to the peptide backbone in SDS micelles but perpendicular in DPC micelles. Further, pKa values of the two histidines change by more than 1 pH unit in SDS relative to DPC, which localizes the imidazole rings to the interfacial region. Line-broadening induced by spin-labelled phosphatidylcholine shows that most of the amino acid side-chains that penetrate the DPC micelle are hydrophobic. Thus, the long axis of the peptide lies parallel to the micelle surface and the hydrophobic face of the alpha-helix provides hydrophobic membrane interaction. The large chemical shift changes shown by Trp11 and N-terminal amino acid residues in SDS relative to DPC indicate that this region may be involved in membrane phospholipid recognition. 1H-NMR assignments, CD spectra, one-dimensional 1H-NMR spectra, chemical-shift analysis and nuclear Overhauser effect information are reported in Supplementary Publication SUP 50184 (11 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K, from whom copies can be obtained according to the terms indicated in Biochem. J. (1997) 321, 8.

A highly mobile C-terminal tail of the Escherichia coli protein export chaperone SecB.

The Escherichia coli export chaperone SecB binds nascent precursors of certain periplasmic and outer membrane proteins and prevents them from folding or aggregating in the cytoplasm. In this study, we demonstrate that the C-terminal 13 residues of SecB were highly mobile using (1)H NMR spectroscopy. A protein lacking the C-terminal 13 amino acids of wild-type SecB was found to retain the ability to bind unfolded maltose-binding protein (MBP) in vitro but to interfere with the normal kinetics of pre-MBP export when overexpressed in vivo. The defect in export was reversed by overproduction of the peripheral membrane ATPase SecA. Therefore, deletion of the mobile region of SecB may alter the interactions of SecB with SecA.

Solution structure of phage lambda half-operator DNA by use of NMR, restrained molecular dynamics, and NOE-based refinement.

The structure of a DNA decamer comprising the left half of the OR3 operator from bacteriophage lambda is determined in solution by using nuclear magnetic resonance spectroscopy and restrained molecular mechanics calculations. Nuclear magnetic resonance assignments for nonexchangeable protons are obtained by two-dimensional correlated and nuclear Overhauser effect (NOE) spectroscopies. Exchangeable proton resonances are assigned by one-dimensional NOE experiments. Coupling constant measurements from one- and two-dimensional experiments are used to determine approximate dihedral angles within the deoxyribose ring. Distances between protons are estimated by extrapolating distances derived from the time-dependent NOE intensities to initial mixing times. The sets of dihedral angles and distances form a basis for structure determination by restrained molecular dynamics. Separate runs start from classical A and from B DNA and converge to essentially identical structures (atomic root mean square difference of 0.8 A). The structures are improved by NOE-based refinement in which observed NOE intensities are compared to those calculated by using a full matrix analysis procedure. Final NOE residual (R) factors were less than 0.19. The resultant structures are generally B type in character, but display local sequence-dependent variations in dihedral angles and in the spatial arrangement of adjacent base pairs. Although the entire structure exhibits a small bend, the central core of the half-operator, which comprises the sequence-specific recognition site for cro repressor, is straight.

Recognition of DNA by GAL4 in solution: use of a monomeric protein-DNA complex for study by NMR.

The complex of a monomer of GAL4 with DNA has been investigated by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. Previous X-ray analysis has revealed a structure in which a dimer of the N-terminal 65-residue fragment of GAL4 forms a complex, 27 kDa in molecular mass, with a 19 base pair full-binding-site DNA [Marmorstein, R., Carey, M., Ptashne, M., & Harrison, S. C. (1992) Nature 356, 408-414]. We have developed a smaller system, half in molecular mass, which is amenable for detailed analysis using NMR. Titration of a 10 base pair half-binding-site DNA with GAL4-(65) shows 1:1 binding, illustrating that one monomer of the protein binds in a specific manner to half-site DNA. The components of the protein-DNA complex are mainly in fast exchange on the NMR chemical shift time scale, with an equilibrium dissociation constant of 161 +/- 12 microM. With a basis of chemical shift data for free GAL4 protein and for the free half-site DNA, the fast exchange facilitates 1H resonance assignments in the complex since cross-peak positions can be examined at different protein:DNA ratios. Chemical shift changes in the DNA reveal the base pairs that are important for recognition by GAL4. Intermolecular NOE cross-peaks are also observed in spectra of the protein-DNA complex. Their identification places the C-terminal end of the first alpha-helix (residues 12-17) in a position such that the amino acids are able to read the DNA sequence in a manner entirely consistent with the X-ray structure of the related complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of the metal-free gamma-carboxyglutamic acid-rich membrane binding region of factor IX by two-dimensional NMR spectroscopy.

The gamma-carboxyglutamic acid-rich domain of blood coagulation Factor IX is required for the binding of the protein to phospholipid membranes. To investigate the three-dimensional structure of this domain, a synthetic peptide corresponding to residues 1-47 of Factor IX was studied by 1H NMR spectroscopy. In the absence of metal ions, the proton chemical shift dispersion in the one-dimensional NMR spectrum indicated that the peptide contains regular structural elements. Upon the addition of Ca(II) or Mg(II), large chemical shift changes were observed in the amide proton and methyl proton regions of the spectrum, consistent with the conformational transitions that metal ions are known to induce in native Factor IX. The apopeptide was studied by two-dimensional NMR spectroscopy at 500 MHz to determine its solution structure. Protons were assigned using total correlation spectroscopy, nuclear Overhauser effect spectroscopy, and double quantum-filtered correlation spectroscopy experiments. Intensities of cross-peaks in the nuclear Overhauser effect spectrum were used to generate a set of interproton distance restraints. The structure of the apopeptide was then calculated using distance geometry methods. There are three structural elements in the apopeptide that are linked by a flexible polypeptide backbone. These elements include a short amino-terminal tetrapeptide loop (amino acids 6-9), the disulfide-containing hexapeptide loop (amino acids 18-23), and a carboxyl-terminal alpha helix (amino acids 37-46). Amide hydrogen exchange kinetics indicate that the majority of the peptide is solvent accessible, except in the carboxyl-terminal element. The structured regions in the apopeptide are insufficient to support phospholipid binding, indicating the importance of additional structural features in the Ca(II)-stabilized conformer.

Solution structure of the DNA-binding domain of Cd2-GAL4 from S. cerevisiae.

The GAL4 protein activates transcription of the genes required for galactose utilization in Saccharomyces cerevisiae. The protein, consisting of 881 amino acids, is dimeric when bound to one of the approximately twofold symmetrical DNA sites present in the galactose upstream activating sequence (UASG). Here we use two-dimensional NMR spectroscopy to determine the structure of an amino-terminal fragment of GAL4 (residues 1-65). This fragment, a monomer in solution, binds as a dimer specifically to UASG-containing DNA. Residues 9-40 form a well defined, compact globular cluster, whereas residues 1-8 and 41-66 show considerable conformational mobility in the absence of DNA. The compact domain contains a motif in which six cysteines, located on two symmetrically related helix/extended strand units connected by a long loop, coordinate two central zinc ions, forming a bimetal-thiolate cluster. The zincs were replaced by NMR-active 113Cd in most of our work and structural parameters are therefore derived from the Cd2-protein. The structure obtained for the GAL4 DNA-binding domain represents a novel DNA-binding motif. Essentially the same conformation is observed for the compact domain in solution using NMR techniques as was seen for the central core of the N-terminal fragment bound to DNA using crystallographic techniques. Thus, the core of the DNA-binding domain changes little upon binding DNA.

Structure of the calcium ion-bound gamma-carboxyglutamic acid-rich domain of factor IX.

We have determined the Ca(II)-bound structure of factor IX, residues 1-47, by nuclear magnetic resonance (NMR) spectroscopy. The amino-terminal 47 residues include the gamma-carboxyglutamic acid-rich and aromatic amino acid stack domains, and this region is responsible for Ca(II)-dependent phospholipid binding in factor IX. Protons in the 1-47 amino acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. A total of 851 distance restraints and 57 torsion angle restraints were used to generate 17 final structures by distance geometry and simulated annealing methods. The backbone RMSD to the geometric average is 0.6 +/- 0.1 A. The Ca(II)-bound structure is substantially more ordered with increased helical content compared to the apo-factor IX (1-47) structure. The global fold is similar to the crystal structure of the Ca(II)-bound Gla domain of prothrombin fragment I from residues 12 to 47 (RMSD approximately 1.3 A), but the backbone conformation differs in the first 11 residues, particularly between residues 3 and 6. The amino-terminal nine Gla residues are oriented to the interior of the protein and suggest an internal Ca(II) binding pocket. The carboxyl-terminal three Gla residues are exposed to solvent. The majority of hydrophobic residues are required to stabilize a globular core in the carboxyl-terminal three-quarters of the molecule. However, a hydrophobic surface patch in the amino-terminal region may represent a phospholipid binding site in factor IX.

Three-dimensional structure of a gamma-carboxyglutamic acid-containing conotoxin, conantokin G, from the marine snail Conus geographus: the metal-free conformer.

Conantokin G is a gamma-carboxyglutamic acid-containing conotoxin from the venom of the marine cone snail Conus geographus. The 17-residue peptide, which contains five gamma-carboxyglutamic acid (Gla) residues and an amidated C-terminal asparagine amide, was synthesized chemically in a form identical to the natural conantokin G. To gain insight into the role of gamma-carboxyglutamic acid in the structure of this peptide, we determined the three-dimensional structure of conantokin G by 1H NMR and compared its structure to other conotoxins and to the gamma-carboxyglutamic acid-containing regions of the vitamin K-dependent blood-clotting proteins. Complete resonance assignments were made by two-dimensional 1H NMR spectroscopy in the absence of metal ions. NOE cross-peaks d(alphaN), d(NN), and d(betaN) provided interproton distance information, and vicinal spin-spin coupling constants 3J(HN alpha) were used to calculate phi torsion angles. Distance geometry and simulated annealing methods were used to derive 20 convergent structures from a set of 227 interproton distance restraints and 13 torsion angle measurements. The backbone rmsd to the geometric average for 20 final structures is 0.8 +/- 0.1 A. Conantokin G consists of a structured region commencing at Gla 3 and extending through arginine 13. This structure includes a partial loop centered around Gla 3 and Gla 4, a distorted type I turn between glutamine 6 and glutamine 9, and two type I turns involving Gla 10, leucine 11, and isoleucine 12 and arginine 13. Together, these two turns define approximately 1.6 turns of a distorted 3(10) helix. The observed structure possesses structural elements similar to those seen in the disulfide-linked conotoxins.