Ubiquitin binding interface mapping on yeast ubiquitin hydrolase by NMR chemical shift perturbation.

The interaction between the 26 kDa yeast ubiquitin hydrolase (YUH1), involved in maintaining the monomeric ubiquitin pool in cells, and the 8.5 kDa yeast ubiquitin protein has been studied by heteronuclear multidimensional NMR spectroscopy. Chemical shift perturbation of backbone (1)H(N), (15)N, and (13)C(alpha) resonances of YUH1, in a YUH1-ubiquitin mixture and in a 35 kDa covalent complex with ubiquitin (a stable analogue of the tetrahedral reaction intermediate), was employed to identify the ubiquitin binding interface of YUH1. This interface mapped on the secondary structure of YUH1 suggests a wide area of contact for ubiquitin, encompassing the N-terminus, alpha1, alpha4, beta2, beta3, and beta6, coincident with the high specificity of YUH1 for ubiquitin. The presence of several hydrophobic clusters in the ubiquitin binding interface of YUH1 suggests that hydrophobic interactions are equally important as ionic interactions in contacting ubiquitin. The residues in the binding interface exhibit a high percentage of homology among the members of the ubiquitin C-terminal hydrolase family, indicating the well-conserved nature of the ubiquitin binding interface reported in this study. The secondary structure of YUH1, from our NMR studies, was similar to the recently determined structure of its human homologue ubiquitin C-terminal hydrolase L3 (UCH-L3), except for the absence of the helix H3 of UCH-L3. This region in YUH1 (helix H3 of UCH-L3) was least perturbed upon ubiquitin binding. Therefore, the binding interface was mapped onto the corresponding residues in the UCH-L3 crystal structure. A model for ubiquitin binding to YUH1 is proposed, in which a good correlation was observed for the lateral binding of ubiquitin to UCH-L3 (YUH1), stabilized by the electrostatic and hydrophobic interactions.     

NMR studies on the substrate-binding domains of the thermosome: structural plasticity in the protrusion region

Group II chaperonins close their cavity with the help of conserved, helical extensions, the so-called protrusions, which emanate from the apical or substrate-binding domains. A comparison of previously solved crystal structures of the apical domains of the thermosome from Thermoplasma acidophilum showed structural plasticity in the protrusion parts induced by extensive packing interactions. In order to assess the influence of the crystal contacts we investigated both the alpha and beta-apical domains (alpha-ADT and beta-ADT) in solution by NMR spectroscopy. Secondary structure assignments and 15N backbone relaxation measurements showed mostly rigid structural elements in the globular parts of the domains, but revealed intrinsic structural disorder and partial helix fraying in the protrusion regions. On the other hand, a beta-turn-motif conserved in archaeal group II chaperonins might facilitate substrate recognition. Our results help us to specify the idea of the open, substrate-accepting state of the thermosome and may provide an additional jigsaw piece in understanding the mode of substrate binding of group II chaperonins.     

Localization of basic residues required for receptor binding to the single alpha-helix of the receptor binding domain of human alpha2-macroglobulin.

To better understand the structural basis for the binding of proteinase-transformed human alpha2-macroglobulin (alpha2M) to its receptor, we have used three-dimensional multinuclear NMR spectroscopy to determine the secondary structure of the receptor binding domain (RBD) of human alpha2M. Assignment of the backbone NMR resonances of RBD was made using 13C/15-N and 15N-enriched RBD expressed in Escherichia coli. The secondary structure of RBD was determined using 1H and 13C chemical shift indices and inter- and intrachain nuclear Overhauser enhancements. The secondary structure consists of eight strands in beta-conformation and one alpha-helix, which together comprise 44% of the protein. The beta-strands form three regions of antiparallel beta-sheet. The two lysines previously identified as being critical for receptor binding are located in (Lys1374), and immediately adjacent to (Lys1370) the alpha-helix, which also contains an (Arg1378). Secondary structure predictions of other alpha-macroglobulins show the conservation of this alpha-helix and suggest an important role for this helix and for basic residues within it for receptor binding.     

Interaction of the E2 and E3 components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Use of a truncated protein domain in NMR spectroscopy

A (15)N-labelled peripheral-subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2p) and the dimer of a solubilized interface domain (E3int) derived from the dihydrolipoyl dehydrogenase (E3) were used to investigate the basis of the interaction of E2p with E3 in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Thirteen of the 55 amino acids in the PSBD show significant changes in either or both of the (15)N and (1)H amide chemical shifts when the PSBD forms a 1 : 1 complex with E3int. All of the 13 amino acids reside near the N-terminus of helix I of PSBD or in the loop region between helix II and helix III. (15)N backbone dynamics experiments on PSBD indicate that the structured region extends from Val129 to Ala168, with limited structure present in residues Asn126 to Arg128. The presence of structure in the region before helix I was confirmed by a refinement of the NMR structure of uncomplexed PSBD. Comparison of the crystal structure of the PSBD bound to E3 with the solution structure of uncomplexed PSBD described here indicates that the PSBD undergoes almost no conformational change upon binding to E3. These studies exemplify and validate the novel use of a solubilized, truncated protein domain in overcoming the limitations of high molecular mass on NMR spectroscopy.     

Mapping the binding of the N-terminal extracellular tail of the CXCR4 receptor to stromal cell-derived factor-1alpha

The solution structure of monomeric stromal cell-derived factor-1alpha (SDF-1alpha), the natural ligand for the CXCR4 G-coupled receptor, has been solved by multidimensional heteronuclear NMR spectroscopy. The structure has a characteristic chemokine fold and is in excellent agreement with the individual subunits observed in the crystal structures of dimeric SDF-1alpha. Using various peptides derived from the N-terminal extracellular tail of the CXCR4 receptor, we show that the principal determinants of binding reside in the N-terminal 17 residues of CXCR4, with a major contribution from the first six residues. From 15N/1HN chemical shift pertubation studies we show that the interaction surface on SDF-1alpha is formed by the undersurface of the three-stranded antiparallel beta-sheet bounded by the N-terminal loop on one side and the C-terminal helix on the other. This surface overlaps with but is not identical to that mapped on several other chemokines for the binding of equivalent peptides derived from their respective receptors.     

A peptide analog of the calmodulin-binding domain of myosin light chain kinase adopts an alpha-helical structure in aqueous trifluoroethanol.

A 22-residue synthetic peptide encompassing the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase was studied by two-dimensional NMR and CD spectroscopy. In water the peptide does not form any regular structure; however, addition of the helix-inducing solvent trifluoroethanol (TFE) causes it to form an alpha-helical structure. The proton NMR spectra of this peptide in 25% and 40% TFE were assigned by double quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser effect correlated spectroscopy spectra. In addition, the alpha-carbon chemical shifts were obtained from (1H,13C)-heteronuclear multiple quantum coherence spectra. The presence of numerous dNN(i, i + 1), d alpha N(i, i + 3), and d alpha beta(i, i + 3) NOE crosspeaks indicates that an alpha-helix can be formed from residues 3 to 20; this is further supported by the CD data. Upfield alpha-proton and downfield alpha-carbon shifts in this region of the peptide provide further support for the formation of an alpha-helix. The helix induced by TFE appears to be similar to that formed upon binding of the peptide to CaM.     

The three-dimensional solution structure and dynamic properties of the human FADD death domain

FADD (also known as MORT-1) is an essential adapter protein that couples the transmembrane receptors Fas (CD95) and tumor necrosis factor receptor-1 (TNF-R1) to intracellular cysteine proteases known as caspases, which propagate and execute the programmed cell death-inducing signal triggered by Fas ligand (FasL, CD95L) and TNF. FADD contains 208 amino acid residues, and comprises two functionally and structurally distinct domains: an N-terminal death effector domain (DED) that promotes activation of the downstream proteolytic cascade through binding of the DED domains of procaspase-8; and a C-terminal death domain (DD). FADD-DD provides the site of FADD recruitment to death receptor complexes at the plasma membrane by, for example, interaction with the Fas receptor cytoplasmic death domain (Fas-DD), or binding of the TNF-R1 adapter molecule TRADD. We have determined the three-dimensional solution structure and characterised the internal polypeptide dynamics of human FADD-DD using heteronuclear NMR spectroscopy of (15)N and (13)C,(15)N-labelled samples. The structure comprises six alpha-helices joined by short loops and displays overall similarity to the death domain of the Fas receptor. The analysis of the dynamic properties reveals no evidence of contiguous stretches of polypeptide chain with increased internal motion, except at the extreme chain termini. A pattern of increased rates of amide proton solvent exchange in the alpha3 helix correlates with a higher degree of solvent exposure for this secondary structure element. The properties of the FADD-DD structure are discussed with respect to previously reported mutagenesis data and emerging models for FasL-induced FADD recruitment to Fas and caspase-8 activation.     

Characterization of the binding interface between the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase

The interaction of the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase, Ccc2a, was investigated by NMR in solution. In particular, a solution of Cu(I)-15NAtx1 was titrated with apo-Ccc2a, and, vice versa, a solution of Cu(I)-15NCcc2a was titrated with apo-Atx1. By following the 15N and 1H chemical shifts, a new species is detected in both experiments. This species is the same in both titrations and is in fast exchange with the parent species on the NMR time scale. Nuclear relaxation data are consistent with the formation of an adduct. Judging from the nuclear Overhauser effect spectroscopy patterns, the structure of Cu(I)-15NCcc2a in the presence of apo-Atx1 is not significantly altered, whereas Cu(I)-15NAtx1 in the presence of apo-Ccc2a experiences some changes with respect to both the apoproteins and the Cu(I)-loaded proteins. The structure of the Cu(I)-15NAtx1 moiety in the adduct was obtained from 1137 nuclear Overhauser effects to a final root mean square deviation to the mean structure of 0.76 +/- 0.13 A for the backbone and 1.11 +/- 0.11 A for the heavy atoms. 15N and 1H chemical shifts suggest the regions of interaction that, together with independent information, allow a structural model of the adduct to be proposed. The apo form of Atx1 displays significant mobility in loops 1 and 5, the N-terminal part of helix alpha1, and the C-terminal part of helix alpha2 on the ms-micros time scale. These regions correspond to the metal binding site. Such mobility is largely reduced in the free Cu(I)-Atx1 and in the adduct with apo-Ccc2a. The analogous mobility of Ccc2a in both Cu(I) and apo forms is reduced with respect to Atx1. Such an adduct is relevant as a structural and kinetic model for copper transfer from Atx1 to Ccc2a in physiological conditions.     

The solution structure of Rhodobacter sphaeroides LH1beta reveals two helical domains separated by a more flexible region: structural consequences for the LH1 complex

Here, the solution structure of the Rhodobacter sphaeroides core light-harvesting complex beta polypeptide solubilised in chloroform:methanol is presented. The structure, determined by homonuclear NMR spectroscopy and distance geometry, comprises two alpha helical regions (residue -34 to -15 and -11 to +6, using the numbering system in which the conserved histidine residue is numbered zero) joined by a more flexible four amino acid residue linker. The C-terminal helix forms the membrane spanning region in the intact LH1 complex, whilst the N-terminal helix must lie in the lipid head groups or in the cytoplasm, and form the basis of interaction with the alpha polypeptide. The structure of a mutant beta polypeptide W(+9)F was also determined. This mutant, which is deficient in a hydrogen bond donor to the bacteriochlorophyll, showed an identical structure to the wild-type, implying that observed differences in interaction with other LH1 polypeptides must arise from cofactor binding. Using these structures we propose a modification to existing models of the intact LH1 complex by replacing the continuous helix of the beta polypeptide with two helices, one of which lies at an acute angle to the membrane plane. We suggest that a key difference between LH1 and LH2 is that the beta subunit is more bent in LH1. This modification puts the N terminus of LH1beta close to the reaction centre H subunit, and provides a rationale for the different ring sizes of LH1 and LH2 complexes.     

Biophysical characterization of elongin C from Saccharomyces cerevisiae

Elongin C (ELC) is an essential component of the mammalian CBC(VHL) E3 ubiquitin ligase complex. As a step toward understanding the role of ELC in assembly and function of CBC-type ubiquitin ligases, we analyzed the quaternary structure and backbone dynamics of the highly homologous Elc1 protein from Saccharomyces cerevisiae. Analytical ultracentrifugation experiments in conjunction with size exclusion chromatography showed that Elc1 is a nonglobular monomer over a wide range of concentrations. Pronounced line broadening in (1)H,(15)N-HSQC NMR spectra and failure to assign peaks corresponding to the carboxy-terminal helix 4 of Elc1 indicated that helix 4 is conformationally labile. Measurement of (15)N NMR relaxation parameters including T(1), T(2), and the (1)H-(15)N nuclear Overhauser effect revealed (i) surprisingly high flexibility of residues 69-77 in loop 5, and (ii) chemical exchange contributions for a large number of residues throughout the protein. Addition of 2,2,2-trifluoroethanol (TFE) stabilized helix 4 and reduced chemical exchange contributions, suggesting that stabilization of helix 4 suppresses the tendency of Elc1 to undergo conformational exchange on a micro- to millisecond time scale. Binding of a peptide representing the major ELC binding site of the von Hippel-Lindau (VHL) tumor suppressor protein almost completely eliminated chemical exchange processes, but induced substantial conformational changes in Elc1 leading to pronounced rotational anisotropy. These results suggest that elongin C interacts with various target proteins including the VHL protein by an induced fit mechanism involving the conformationally flexible carboxy-terminal helix 4.     

Structure of the O-specific polysaccharide of Providencia rustigianii O14 containing N(epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine

The O-specific polysaccharide of Providencia rustigianii O14 was obtained by mild acid degradation of the LPS and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments. The polysaccharide was found to contain N (epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine ('alaninolysine', 2S,8S-AlaLys). The amino acid component was isolated by acid hydrolysis and identified by 13C NMR spectroscopy and specific optical rotation, using synthetic diastereomers for comparison. The following structure of the trisaccharide repeating unit of the polysaccharide was established:Anti-P. rustigianii O14 serum was found to cross-react with O-specific polysaccharides of Providencia and Proteus strains that contains amides of uronic acid with N(epsilon)-[(R)-1-carboxyethyl]-L-lysine and L-lysine.     

Solution structure of human BCL-w: modulation of ligand binding by the C-terminal helix

The structure of human BCL-w, an anti-apoptotic member of the BCL-2 family, was determined by triple-resonance NMR spectroscopy and molecular modeling. Introduction of a single amino acid substitution (P117V) significantly improved the quality of the NMR spectra obtained. The cytosolic domain of BCL-w consists of 8 alpha-helices, which adopt a fold similar to that of BCL-xL, BCL-2, and BAX proteins. Pairwise root meant square deviation values were less than 3 A for backbone atoms of structurally equivalent regions. Interestingly, the C-terminal helix alpha8 of BCL-w folds into the BH3-binding hydrophobic cleft of the protein, in a fashion similar to the C-terminal transmembrane helix of BAX. A peptide corresponding to the BH3 region of the pro-apoptotic protein, BID, could displace helix alpha8 from the BCL-w cleft, resulting in helix unfolding. Deletion of helix alpha8 increased binding affinities of BCL-w for BAK and BID BH3-peptides, indicating that this helix competes for peptide binding to the hydrophobic cleft. These results suggest that although the cytosolic domain of BCL-w exhibits an overall structure similar to that of BCL-xL and BCL-2, the unique organization of its C-terminal helix may modulate BCL-w interactions with pro-apoptotic binding partners.     

Two-dimensional NMR, circular dichroism, and fluorescence studies of PP-50, a synthetic ATP-binding peptide from the beta-subunit of mitochondrial ATP synthase.

PP-50, a peptide based on residues 141-190 of the beta-subunit of mitochondrial F1-ATPase, contains the GX4GKT consensus region for nucleoside triphosphate binding and has been shown to bind ATP [Garboczi, D.N., Shenbagamurthi, W.K., Hullihen, J., & Pedersen, P.L. (1988) J. Biol. Chem. 263, 812-816]. At pH 4.0, appropriate for NMR studies, PP-50 retains the ability to bind ATP tightly (KD = 17.5 microM) with a 1:1 stoichiometry as shown by titrations measuring the partial quenching of ATP fluorescence by PP-50. CD spectra of PP-50 at pH 4.0 and at low ionic strength show 5.8% helix, 30.2% beta-structure, and 64% coil. ATP binding increases the structure of PP-50, changing the CD to 7.5% helix, 44.5% beta-structure, and 48% coil. Increasing the ionic strength to 50 mM KCl also increases the structure, changing the CD to 7.4% helix, 64.4% beta-structure, and 28.2% coil. The 600-MHz proton NMR spectrum of PP-50, at pH 4.0 and low ionic strength, has been assigned by 2D methods (TOCSY, DQF-COSY, and NOESY with jump-return water suppression). Based on strong d alpha N NOEs, J alpha N values, and NH chemical shifts differing from random coil values, regions of extended structure are detected from residues 1-7 and 43-48. Based on dNN, dNN(i,i+2), and d alpha N(i,i+2) NOEs and 3J alpha N values, possible type I' and type I turns are found from residues 11-14 and 31-34, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The unfolding of oxidized c-type cytochromes: the instructive case of Bacillus pasteurii

The reversible unfolding of oxidized Bacillus pasteurii cytochrome c(553) by guanidinium chloride under equilibrium conditions has been monitored by NMR and optical spectroscopy. The results obtained indicate that unfolding takes place through a mechanism involving the detachment from heme iron coordination of the sulfur of the Met71 axial ligand and yielding either a high spin (HS) or a low spin (LS(1)) species, depending on the pH value. In the LS(1) form the Met71 is replaced by another protein ligand, possibly Lys. The ligand exchange reaction does not reach completion until the protein backbone reaches a largely unfolded state, as monitored through 1H-15N NMR experiments, thus demonstrating that there is a significant correlation between formation of the Fe-S bond and native structure stability. 1H/2H exchange data, however, show that helix alpha(3), the C-terminal region of helix alpha(4), and helix alpha(5) maintain low exchangeability of the amide protons in the LS(1) form. This finding most likely implies that these regions maintain some ordered non-covalent structure, in which the amide moieties are involved in H-bonds. Finally, a folding mechanism is proposed and discussed in terms of analogies and differences with the larger mitochondrial cytochrome c proteins. It is concluded that the thermodynamic stability of the region around the metal cofactor is determined by the chemical nature of the residues around the axial methionine residue.     

Elucidation of the epsilon-theta subunit interface of Escherichia coli DNA polymerase III by NMR spectroscopy

The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli. The epsilon (epsilon) subunit of HE provides the 3'-->5' exonucleolytic proofreading activity for this complex. Epsilon consists of two domains: an N-terminal domain containing the proofreading exonuclease activity (residues 1-186) and a C-terminal domain required for binding to the polymerase (alpha) subunit (residues 187-243). In addition to alpha, epsilon also binds the small (8 kDa) theta (theta) subunit. The function of theta is unknown, although it has been hypothesized to enhance the 3'-->5' exonucleolytic proofreading activity of epsilon. Using NMR analysis and molecular modeling, we have previously reported a structural model of epsilon186, the N-terminal catalytic domain of epsilon [DeRose et al. (2002) Biochemistry 41, 94]. Here, we have performed 3D triple resonance NMR experiments to assign the backbone and C(beta) resonances of [U-(2)H,(13)C,(15)N] methyl protonated epsilon186 in complex with unlabeled theta. A structural comparison of the epsilon186-theta complex with free epsilon186 revealed no major changes in secondary structure, implying that the overall structure is not significantly perturbed in the complex. Amide chemical shift comparisons between bound and unbound epsilon186 revealed a potential binding surface on epsilon for interaction with theta involving structural elements near the epsilon catalytic site. The most significant shifts observed for the epsilon186 amide resonances are localized to helix alpha1 and beta-strands 2 and 3 and to the region near the beginning of alpha-helix 7. Additionally, a small stretch of residues (K158-L161), which previously had not been assigned in uncomplexed epsilon186, is predicted to adopt beta-strand secondary structure in the epsilon186-theta complex and may be significant for interaction with theta. The amide shift pattern was confirmed by the shifts of aliphatic methyl protons, for which the larger shifts generally were concentrated in the same regions of the protein. These chemical shift mapping results also suggest an explanation for how the unstable dnaQ49 mutator phenotype of epsilon may be stabilized by binding theta.     

Solution structure of the single-domain prolyl cis/trans isomerase PIN1At from Arabidopsis thaliana

The 119-amino acid residue prolyl cis/trans isomerase from Arabidopsis thaliana (PIN1At) is similar to the catalytic domain of the human hPIN1. However, PIN1At lacks the N-terminal WW domain that appears to be essential for the hPIN1 function. Here, the solution structure of PIN1At was determined by three-dimensional nuclear magnetic resonance spectroscopy. The PIN1At fold could be superimposed on that of the catalytic domain of hPIN1 and had a 19 residue flexible loop located between strand beta1 and helix alpha1. The dynamical features of this beta1/alpha1-loop, which are characteristic for a region involved in protein-protein interactions, led to exchange broadening in the NMR spectra. When sodium sulfate salt was added to the protein sample, the beta1/alpha1 loop was stabilized and, hence, a complete backbone resonance assignment was obtained. Previously, with a phospho-Cdc25 peptide as substrate, PIN1At had been shown to catalyze the phosphoserine/phosphothreonine prolyl cis/trans isomerization specifically. To map the catalytic site of PIN1At, the phospho-Cdc25 peptide or sodium sulfate salt was added in excess to the protein and chemical shift changes in the backbone amide protons were monitored in the (1)H(N)-(15)N heteronuclear single quantum coherence spectrum. The peptide caused perturbations in the loops between helix alpha4 and strand beta3, between strands beta3 and beta4, in the alpha3 helix, and in the beta1/alpha1 loop. The amide groups of the residues Arg21 and Arg22 showed large chemical shift perturbations upon phospho-Cdc25 peptide or sulfate addition. We conclude that this basic cluster formed by Arg21 and Arg22, both located in the beta1/alpha1 loop, is homologous to that found in the hPIN1 crystal structure (Arg68 and Arg69), which also is involved in sulfate ion binding. We showed that the sulfate group competed for the interaction between PIN1At and the phospho-Cdc25 peptide. In the absence of the WW domain, three hydrophobic residues (Ile33, Ile34, and Leu35) located in the long flexible loop and specific for the plant PIN-type peptidyl prolyl cis/trans isomerases (PPIases) could be an additional interaction site in PIN1At. However, phospho-peptide addition did not affect the resonances of these residues significantly. Electrostatic potential calculations revealed a negatively charged area not found in hPIN1 on the PIN1At molecular surface, which corresponds to the surface shielded by the WW domain in hPIN1. Based on our experimental results and the molecular specificities of the PIN1At enzyme, functional implications of the lack of WW domains in this plant PIN-type PPIase will be discussed.     

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.     

1H NMR and CD secondary structure analysis of cell adhesion promoting peptide F-9 from laminin

A heparin binding, cell adhesion promoting domain, termed peptide F-9, from the B1 chain of human laminin, residues 641 to 660, i.e. RYVVLPRPVCFEKGMNYTVR, has been investigated by 1H NMR (500 MHz) spectroscopy and CD spectropolarimetry. While small linear peptides in water solution normally exist in a number of fluctuating conformational states, CD data analysis of peptide F9 indicates the existence of some preferred average structural populations consisting of about 30% beta-sheet, 22% beta-turn, and 6% alpha-helix. NMR structural analysis supports this observation and indicates specific sequences of preferred structural populations. Evidence for these is indicated by the presence of dNN nuclear Overhauser effect (NOE) populations and attenuated or absent d alpha N NOEs at short mixing times (0.1 s), 3J alpha N coupling constants of 5 and 10 Hz, and chemical shifts significantly removed from random coil positions. The NH2-terminal VVL sequence primarily exists in an extended chain conformation by virtue of large d alpha N NOEs and 9-10 Hz 3J alpha N coupling constants. Residues C10-N16 have turn-like or helix character with a run of dNN and d beta N NOEs and attenuated d alpha N NOEs. These midchain reversals include the lysine and asparagine residues proposed to be involved in heparin binding and N-glycosylation, respectively, to laminin peptide F-9.