Fast atom bombardment mass spectrometry of bleomycin A2 and B2 and their metal complexes

The new mass spectrometric technique of fast atom bombardment is applied to the analysis of bleomycins either separately or in mixtures. The spectra are reproducible and afford valuable analytical data and structurally significant fragmentation patterns on these important antibiotics. The procedures outlined have also enabled the characterisation of various metal complexes.     

The folding of an enzyme. V. H/2H exchange-nuclear magnetic resonance studies on the folding pathway of barnase: complementarity to and agreement with protein engineering studies.

Two major methods are currently being used to characterize transient intermediates during protein folding at the level of individual residues. Nuclear magnetic resonance (n.m.r.) measurements on the protection of peptide NH hydrogens against exchange with solvent during refolding can provide information about secondary structure formation. Protein engineering and kinetics can provide direct information about intramolecular interactions of protein side-chains and indirect evidence on secondary structure. These procedures have provided the most complete pictures so far about protein folding intermediates. Both methods have been applied to the characterization of an intermediate in the refolding of barnase. Although the two methods give complementary information, there are some regions of the protein where the methods overlap well. We show that, with one possible exception that is obscure, n.m.r. and protein engineering give identical results for those interactions that can be analysed by both methods. This suggests that these are valid approaches for the study of protein folding intermediates in the case of barnase and that the combination of the methods is a powerful analytical procedure. Information provided by n.m.r. data that is complementary to the protein engineering experiments is: (1) early formation of the C terminus of helix2; (2) early formation of helix3; (3) early formation of several beta-turns (46-49, 101-104 in loop5); and (5) partial formation of loop5. Confirmatory evidence of protein engineering data on the intermediate is: (1) helix1 is complete from residues 10 to 18; (2) the interactions between all beta-strands are present; (3) part of loop2 is not formed; (4) part of loop3 is formed; and (5) some specific tertiary interactions are not made. For some interactions the protein engineering and H/2H exchange methods overlap directly. The information obtained for direct overlap is self consistent.     

Role of conformational heterogeneity in domain swapping and adapter function of the Cks proteins

Cks proteins are adapter molecules that coordinate the assembly of multiprotein complexes. They share the ability to domain swap by exchanging a beta-strand, beta4. Here we use NMR spectroscopy and molecular dynamics simulations to investigate the dynamic properties of human Cks1 and its response on assembly with components of the SCF(Skp2) ubiquitin ligation machinery. In the NMR experiment with the free form of Cks1, a subset of residues displayed elevated R2 values and the cross-peaks of neighboring residues were missing from the spectrum, indicating a substantial conformational exchange contribution on the microsecond to millisecond time scale. Strikingly the region of greatest conformational variability was the beta4-strand that domain swaps to form the dimer. Binding of the ligand common to all Cks proteins, Cdk2, suppressed the conformational heterogeneity. This response was specific to Cdk2 binding; in contrast, binding of Skp2, a ligand unique to human Cks1, did not alter the dynamic behavior. Short time (<5 ns) molecular dynamics simulations indicate that residues of Cks1 that form the binding site for phosphorylated ligands are considerably more flexible in the free form of Cks1 than they are in the Cdk2-Cks1 complex. A cooperative interaction between Cdk2 and Cks1 is suggested, which reduces the configurational entropy of Cks1 and therefore facilitates phosphoprotein binding. Indications of an unusual dynamic behavior of strand beta4 in the free form of Cks1 were obtained from longer time scale (50 ns) dynamics simulations. A spontaneous reversible unzipping of hydrogen bonds between beta4 and beta2 was observed, suggesting an early intermediate structure for unfolding and/or domain swapping. We propose that the dynamic properties of the beta-sheet and its modification upon ligand binding underlie the domain swapping ability and the adapter function of Cks proteins.     

The structure of a LysM domain from E. coli membrane-bound lytic murein transglycosylase D (MltD)

The LysM domain is a widespread protein module. It was originally identified in enzymes that degrade bacterial cell walls but is also present in many other bacterial proteins. Several proteins that contain the domain, such as Staphylococcal IgG binding proteins and Escherichia coli intimin, are involved in bacterial pathogenesis. LysM domains are also found in some eukaryotic proteins, apparently as a result of horizontal gene transfer from bacteria. The available evidence suggests that the LysM domain is a general peptidoglycan-binding module. We have determined the structure of this domain from E. coli membrane-bound lytic murein transglycosylase D. The LysM domain has a betaalphaalphabeta secondary structure with the two helices packing onto the same side of an anti- parallel beta sheet. The structure shows no similarity to other bacterial cell surface domains. A potential binding site in a shallow groove on surface of the protein has been identified.     

The UBX domain: a widespread ubiquitin-like module

The UBX domain is an 80 amino acid residue module that is present typically at the carboxyl terminus of a variety of eukaryotic proteins. In an effort to elucidate the function of UBX domains, we solved the three-dimensional structure of the UBX domain of human Fas-associated factor-1 (FAF1) by NMR spectroscopy. The structure has a beta-Grasp fold characterised by a beta-beta-alpha-beta-beta-alpha-beta secondary-structure organisation. The five beta strands are arranged into a mixed sheet in the order 21534. The longer first helix packs across the first three strands of the sheet, and a second shorter 3(10) helix is located in an extended loop connecting strands 4 and 5. In the absence of significant sequence similarity, the UBX domain can be superimposed with ubiquitin with an r.m.s.d. of 1.9 A, suggesting that the two structures share the same superfold, and an evolutionary relationship. However, the absence of a carboxyl-terminal extension containing a double glycine motif and of suitably positioned lysine side-chains makes it highly unlikely that UBX domains are either conjugated to other proteins or part of mixed UBX-ubiquitin chains. Database searches revealed that most UBX domain-containing proteins belong to one of four evolutionarily conserved families represented by the human FAF1, p47, Y33K, and Rep8 proteins. A role of the UBX domain in ubiquitin-related processes is suggested.     

The ligand-binding loops in the tunicate C-type lectin TC14 are rigid

C-type lectin-like domains are very common components of extracellular proteins in animals. They bind to a variety of ligands, including carbohydrates, proteins, ice, and CaCO3 crystals. Their structure is characterized by long surface loops in the area of the protein usually involved in ligand binding. The C-type lectin TC14 from Polyandrocarpa misakiensis specifically binds to D-galactose by coordination of the sugar to a bound calcium atom. We have studied the dynamic properties of TC14 by measuring 15N longitudinal and transverse relaxation rates as well as [1H-15N] heteronuclear NOEs. Relaxation rates and heteronuclear NOE data for holo-TC14 show minimal variations, indicating that there is no substantial difference in rigidity between the elements of regular secondary structure and the extended surface loops. Anisotropic tumbling of the elongated TC14 dimer can account for the main fluctuations in relaxation rates. Loss of the bound calcium does not significantly alter the internal dynamics, suggesting that the stability of the loop region is intrinsic and not dependent on the coordination of the calcium ion. Chemical shift differences between the holo and apo form show that main structural changes occur in the calcium-binding site, but smaller structural changes are propagated throughout the molecule without affecting the overall fold. The disappearance of two resonances for residues following the conserved cis-proline 87 (which is located in the calcium-binding site) in the apo form indicates conformational change on an NMR time scale between the cis and trans configurations of this peptide bond in the absence of calcium. Possible implications of these findings for the ligand binding in C-type lectin-like domains are discussed.