Membrane glycoprotein synthesis: cleavage of the signal sequence in the absence of glycosylation

The controlled action of trypsin on porcine pancreatic procarboxypeptidase A releases a large activation peptide which contains the activation segment of the proenzyme. Circular dichroism studies indicate that the isolated activation peptide contains a high percentage of residues in ordered secondary structures (mainly α-helix). This result agrees with predictions of secondary structure carried out on the published amino acid sequence of the homologous rat proenzyme. Moreover, proton magnetic resonance spectroscopy shows that the peptide adopts a thermostable tertiary structure with characteristics typical of globular proteins. The results as a whole indicate that the activation segment of porcine pancreatic procarboxypeptidase A constitutes a folded structural domain.     

Nuclear magnetic resonance solution structure of hirudin(1-51) and comparison with corresponding three-dimensional structures determined using the complete 65-residue hirudin polypeptide chain.

The three-dimensional structure of the N-terminal 51-residue domain of recombinant hirudin in aqueous solution was determined by 1H nuclear magnetic resonance (NMR) spectroscopy, and the resulting high-quality solution structure was compared with corresponding structures obtained from studies with the intact, 65-residue polypeptide chain of hirudin. On the basis of 580 distance constraints derived from nuclear Overhauser effects and 109 dihedral angle constraints, a group of 20 conformers representing the solution structure of hirudin(1-51) was computed with the program DIANA and energy-minimized with a modified version of the program AMBER. Residues 3 to 30 and 37 to 48 form a well-defined molecular core with two antiparallel beta-sheets composed of residues 14 to 16 and 20 to 22, and 27 to 31 and 36 to 40, and three reverse turns at residues 8 to 11 (type II), 17 to 20 (type II') and 23 to 26 (type II). The average root-mean-square deviation of the individual NMR conformers relative to their mean co-ordinates is 0.38 A for the backbone atoms and 0.77 A for all heavy atoms of these residues. Increased structural disorder was found for the N-terminal dipeptide segment, the loop at residues 31 to 36, and the C-terminal tripeptide segment. The solution structure of hirudin(1-51) has the same molecular architecture as the corresponding polypeptide segment in natural hirudin and recombinant desulfatohirudin. It is also closely similar to the crystal structure of the N-terminal 51-residue segment of hirudin in a hirudin-thrombin complex, with root-mean-square deviations of the crystal structure relative to the mean solution structure of 0.61 A for the backbone atoms and 0.91 A for all heavy atoms of residues 3 to 30 and 37 to 48. Further coincidence is found for the loop formed by residues 31 to 36, which shows increased structural disorder in all available solution structures of hirudin, and of which residues 32 to 35 are not observable in the electron density map of the thrombin complex. Significant local structural differences between hirudin(1-51) in solution and hirudin in the crystalline thrombin complex were identified mainly for the N-terminal tripeptide segment and residues 17 to 21. These are further analyzed in an accompanying paper.     

Structure discrimination for the C-terminal domain of Escherichia coli trigger factor in solution

NMR measurements can give important information on solution structure, without the necessity for a full-scale solution structure determination. The C-terminal protein binding domain of the ribosome-associated chaperone protein trigger factor is composed of non-contiguous parts of the polypeptide chain, with an interpolated prolyl isomerase domain. A construct of the C-terminal domain of Escherichia coli trigger factor containing residues 113–149 and 247–432, joined by a Gly-Ser-Gly-Ser linker, is well folded and gives excellent NMR spectra in solution. We have used NMR measurements on this construct, and on a longer construct that includes the prolyl isomerase domain, to distinguish between two possible structures for the C-terminal domain of trigger factor, and to assess the behavior of the trigger factor C-terminal domain in solution. Two X-ray crystal structures, of intact trigger factor from E. coli (Ferbitz et al., Nature 431:590–596, 2004), and of a truncated trigger factor from Vibrio cholerae (Ludlam et al., Proc Natl Acad Sci USA 101:13436–13441, 2004) showed significant differences in the structure of the C-terminal domain, such that the two structures could not be superimposed. We show using NMR chemical shifts and long range nuclear Overhauser effects that the secondary and tertiary structure of the E. coli C-terminal domain in solution is consistent with the crystal structure of the E. coli trigger factor and not with the V. cholerae protein. Given the similarity of the amino acid sequences of the E. coli and V. cholerae proteins, it appears likely that the structure of the V. cholerae protein has been distorted as a result of truncation of a 44-amino acid segment at the C-terminus. Analysis of residual dipolar coupling measurements shows that the overall topology of the solution structure is completely inconsistent with both structures. Dynamics analysis of the C-terminal domain using T₁, T₂ and heteronuclear NOE parameters show that the protein is overall rather flexible. These results indicate that the structure of this domain in solution resembles the X-ray crystal structure of the E. coli protein in secondary structure and at least some tertiary contacts, but that the overall topology differs in solution, probably due to structural fluctuation.     

Secondary structure and dynamics of an intrinsically unstructured linker domain

The transient secondary structure and dynamics of an intrinsically unstructured linker domain from the 70 kDa subunit of human replication protein A was investigated using solution state NMR. Stable secondary structure, inferred from large secondary chemical shifts, was observed for a segment of the intrinsically unstructured linker domain when it is attached to an N-terminal protein interaction domain. Results from NMR relaxation experiments showed the rotational diffusion for this segment of the intrinsically unstructured linker domain to be correlated with the N-terminal protein interaction domain. When the N-terminal domain is removed, the stable secondary structure is lost and faster rotational diffusion is observed. The large secondary chemical shifts were used to calculate phi and psi dihedral angles and these dihedral angles were used to build a backbone structural model. Restrained molecular dynamics were performed on this new structure using the chemical shift based dihedral angles and a single NOE distance as restraints. In the resulting family of structures a large, solvent exposed loop was observed for the segment of the intrinsically unstructured linker domain that had large secondary chemical shifts.     

Crystal structure of fervidolysin from Fervidobacterium pennivorans, a keratinolytic enzyme related to subtilisin

Structure-forming fibrous proteins like keratins, gelatins and collagens are degraded only by a few proteases as their tight packing limits access to the potential cleavage sites. To understand the keratin degradation in detail, we describe the first crystal structure of a keratin-degrading enzyme (keratinase), fervidolysin, from Fervidobacterium pennivorans as an immature form with propeptide (PD)-bound. The 1.7A resolution crystal structure shows that the protease is composed of four domains: a catalytic domain (CD), two beta-sandwich domains (SDs), and the PD domain. A structural alignment shows a distant relationship between the PD-CD substructure of fervidolysin and pro-subtilisin E. Tight binding of PD to the remaining part of the protease is mediated by hydrogen bonds along the domain surfaces and around the active cleft, and by the clamps to SD1 and SD2. The crystal structure of this multi-domain protein fervidolysin provides insights into proenzyme activation and the role of non-catalytic domains, suggesting a functional relationship to the fibronectin (FN)-like domains of the human promatrix metalloprotease-2 (proMMP-2) that degrades the fibrous polymeric substrate gelatin.     

Solution structure of (gamma)S-crystallin by molecular fragment replacement NMR

The solution structure of murine gammaS-crystallin (gammaS) has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of dipolar couplings, recorded in different alignment media, and supplemented by a small number of NOE distance restraints. gammaS consists of two topologically similar domains, arranged with an approximate twofold symmetry, and each domain shows close structural homology to closely related (approximately 50% sequence identity) domains found in other members of the gamma-crystallin family. Each domain consists of two four-strand "Greek key" beta-sheets. Although the domains are tightly anchored to one another by the hydrophobic surfaces of the two inner Greek key motifs, the N-arm, the interdomain linker and several turn regions show unexpected flexibility and disorder in solution. This may contribute entropic stabilization to the protein in solution, but may also indicate nucleation sites for unfolding or other structural transitions. The method used for solving the gammaS structure relies on the recently introduced molecular fragment replacement method, which capitalizes on the large database of protein structures previously solved by X-ray crystallography and NMR.     

The Three-Dimensional Solution Structure of the Src Homology Domain-2 of the Growth Factor Receptor-Bound Protein-2

A set of high-resolution three-dimensional solution structures of the Src homology region-2 (SH2) domain of the growth factor receptor-bound protein-2 was determined using heteronuclear NMR spectroscopy. The NMR data used in this study were collected on a stable monomeric protein solution that was free of protein aggregates and proteolysis. The solution structure was determined based upon a total of 1439 constraints, which included 1326 nuclear Overhauser effect distance constraints, 70 hydrogen bond constraints, and 43 dihedral angle constraints. Distance geometry-simulated annealing calculations followed by energy minimization yielded a family of 18 structures that converged to a root-mean-square deviation of 1.09 Å for all backbone atoms and 0.40 Å for the backbone atoms of the central -sheet. The core structure of the SH2 domain contains an antiparallel -sheet flanked by two parallel -helices displaying an overall architecture that is similar to other known SH2 domain structures. This family of NMR structures is compared to the X-ray structure and to another family of NMR solution structures determined under different solution conditions.     

Solution structure of inhibitor-free human metalloelastase (MMP-12) indicates an internal conformational adjustment

Macrophage metalloelastase or matrix metalloproteinase-12 (MMP-12) appears to exacerbate atherosclerosis, emphysema, aortic aneurysm, rheumatoid arthritis, and inflammatory bowel disease. An inactivating E219A mutation, validated by crystallography and NMR spectra, prevents autolysis of MMP-12 and allows us to determine its NMR structure without an inhibitor. The structural ensemble of the catalytic domain without an inhibitor is based on 2813 nuclear Overhauser effects (NOEs) and has an average RMSD to the mean structure of 0.25 Å for the backbone and 0.61 Å for all heavy atoms for residues Trp109-Gly263. Compared to crystal structures of MMP-12, helix B (hB) at the active site is unexpectedly more deeply recessed under the β-sheet. This opens a pocket between hB and β-strand IV in the active-site cleft. Both hB and an internal cavity are shifted toward β-strand I, β-strand III, and helix A on the back side of the protease. About 25 internal NOE contacts distinguish the inhibitor-free solution structure and indicate hB's greater depth and proximity to the sheet and helix A. Line broadening and multiplicity of amide proton NMR peaks from hB are consistent with hB undergoing a slow conformational exchange among subtly different environments. Inhibitor-binding-induced perturbations of the NMR spectra of MMP-1 and MMP-3 map to similar locations across MMP-12 and encompass the internal conformational adjustments. Evolutionary trace analysis suggests a functionally important network of residues that encompasses most of the locations adjusting in conformation, including 18 residues with NOE contacts unique to inhibitor-free MMP-12. The conformational change, sequence analysis, and inhibitor perturbations of NMR spectra agree on the network they identify between structural scaffold and the active site of MMPs.     

Conformational analysis of peptides corresponding to all the secondary structure elements of protein L B1 domain: secondary structure propensities are not conserved in proteins with the same fold.

The solution conformation of three peptides corresponding to the two beta-hairpins and the alpha-helix of the protein L B1 domain have been analyzed by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). In aqueous solution, the three peptides show low populations of native and non-native locally folded structures, but no well-defined hairpin or helix structures are formed. In 30% aqueous trifluoroethanol (TFE), the peptide corresponding to the alpha-helix adopts a high populated helical conformation three residues longer than in the protein. The hairpin peptides aggregate in TFE, and no significant conformational change occurs in the NMR observable fraction of molecules. These results indicate that the helical peptide has a significant intrinsic tendency to adopt its native structure and that the hairpin sequences seem to be selected as non-helical. This suggests that these sequences favor the structure finally attained in the protein, but the contribution of the local interactions alone is not enough to drive the formation of a detectable population of native secondary structures. This pattern of secondary structure tendencies is different to those observed in two structurally related proteins: ubiquitin and the protein G B1 domain. The only common feature is a certain propensity of the helical segments to form the native structure. These results indicate that for a protein to fold, there is no need for large native-like secondary structure propensities, although a minimum tendency to avoid non-native structures and to favor native ones could be required.     

High resolution solution structure of apo calcyclin and structural variations in the S100 family of calcium-binding proteins

The three-dimensional solution structure of apo rabbit lung calcyclin has been refined to high resolution through the use of heteronuclear NMR spectroscopy and 13C,15N- enriched protein. Upon completing the assignment of virtually all of the 15N, 13C and 1H NMR resonances, the solution structure was determined from a combination of 2814 NOE- derived distance constraints, and 272 torsion angle constraints derived from scalar couplings. A large number of critical inter- subunit NOEs (386) were identified from 13C- select,13C-filtered NOESY experiments, providing a highly accurate dimer interface. The combination of distance geometry and restrained molecular dynamics calculations yielded structures with excellent agreement with the experimental data and high precision (rmsd from the mean for the backbone atoms in the eight helices: 0.33 Å). Calcyclin exhibits a symmetric dimeric fold of two identical 90 amino acid subunits, characteristic of the S100 subfamily of EF-hand Ca2+-binding proteins. The structure reveals a readily identified pair of putative sites for binding of Zn2+. In order to accurately determine the structural features that differentiate the various S100 proteins, distance difference matrices and contact maps were calculated for the NMR structural ensembles of apo calcyclin and rat and bovine S100B. These data show that the most significant variations among the structures are in the positioning of helix III and in loops, the regions with least sequence similarity. Inter-helical angles and distance differences for the proteins show that the positioning of helix III of calcyclin is most similar to that of bovine S100B, but that the helix interfaces are more closely packed in calcyclin than in either S100B structure. Surprisingly large differences were found in the positioning of helix III in the two S100B structures, despite there being only four non-identical residues, suggesting that one or both of the S100B structures requires further refinement.     

Solution structure of the helicase-interaction domain of the primase DnaG: a model for helicase activation

The helicase-primase interaction is a critical event in DNA replication and is mediated by a putative helicase-interaction domain within the primase. The solution structure of the helicase-interaction domain of DnaG reveals that it is made up of two independent subdomains: an N-terminal six-helix module and a C-terminal two-helix module that contains the residues of the primase previously identified as important in the interaction with the helicase. We show that the two-helix module alone is sufficient for strong binding between the primase and the helicase but fails to activate the helicase; both subdomains are required for helicase activation. The six-helix module of the primase has only one close structural homolog, the N-terminal domain of the corresponding helicase. This surprising structural relationship, coupled with the differences in surface properties of the two molecules, suggests how the helicase-interaction domain may perturb the structure of the helicase and lead to activation.     

Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.     

NMR structure of a KlbA intein precursor from Methanococcus jannaschii

Certain proteins of unicellular organisms are translated as precursor polypeptides containing inteins (intervening proteins), which are domains capable of performing protein splicing. These domains, in conjunction with a single residue following the intein, catalyze their own excision from the surrounding protein (extein) in a multistep reaction involving the cleavage of two intein-extein peptide bonds and the formation of a new peptide bond that ligates the two exteins to yield the mature protein. We report here the solution NMR structure of a 186-residue precursor of the KlbA intein from Methanococcus jannaschii, comprising the intein together with N- and C-extein segments of 7 and 11 residues, respectively. The intein is shown to adopt a single, well-defined globular domain, representing a HINT (Hedgehog/Intein)-type topology. Fourteen beta-strands are arranged in a complex fold that includes four beta-hairpins and an antiparallel beta-ribbon, and there is one alpha-helix, which is packed against the beta-ribbon, and one turn of 3(10)-helix in the loop between the beta-strands 8 and 9. The two extein segments show increased disorder, and form only minimal nonbonding contacts with the intein domain. Structure-based mutation experiments resulted in a proposal for functional roles of individual residues in the intein catalytic mechanism.     

NMR solution structure of the RED subdomain of the Sleeping Beauty transposase

DNA transposons can be employed for stable gene transfer in vertebrates. The Sleeping Beauty (SB) DNA transposon has been recently adapted for human application and is being evaluated in clinical trials, however its molecular mechanism is not clear. SB transposition is catalyzed by the transposase enzyme, which is a multi‐domain protein containing the catalytic and the DNA‐binding domains. The DNA‐binding domain of the SB transposase contains two structurally independent subdomains, PAI and RED. Recently, the structures of the catalytic domain and the PAI subdomain have been determined, however no structural information on the RED subdomain and its interactions with DNA has been available. Here, we used NMR spectroscopy to determine the solution structure of the RED subdomain and characterize its interactions with the transposon DNA.     

A change in conformational dynamics underlies the activation of Eph receptor tyrosine kinases

Eph receptor tyrosine kinases (RTKs) mediate numerous developmental processes. Their activity is regulated by auto-phosphorylation on two tyrosines within the juxtamembrane segment (JMS) immediately N-terminal to the kinase domain (KD). Here, we probe the molecular details of Eph kinase activation through mutational analysis, X-ray crystallography and NMR spectroscopy on auto-inhibited and active EphB2 and EphA4 fragments. We show that a Tyr750Ala gain-of-function mutation in the KD and JMS phosphorylation independently induce disorder of the JMS and its dissociation from the KD. Our X-ray analyses demonstrate that this occurs without major conformational changes to the KD and with only partial ordering of the KD activation segment. However, conformational exchange for helix alphaC in the N-terminal KD lobe and for the activation segment, coupled with increased inter-lobe dynamics, is observed upon kinase activation in our NMR analyses. Overall, our results suggest that a change in inter-lobe dynamics and the sampling of catalytically competent conformations for helix alphaC and the activation segment rather than a transition to a static active conformation underlies Eph RTK activation.