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Torbjörn Drakenberg

Publications and source records attributed to Torbjörn Drakenberg.

11 recordsLinked to original sources
PubMed2006

Solution structures of human and porcine beta-microseminoprotein.

Beta-microseminoprotein (MSP) is a small cysteine-rich protein (molecular mass about 10 kDa) first isolated from human seminal plasma and later identified in several other organisms. The function of MSP is not known, but a recent study has shown MSP to bind CRISP-3, a protein present in neutrophilic granulocytes. The amino acid sequence is highly variable between species raising the question of the evolutionary conservation of the 3D structure. Here we present NMR solution structures of both the human and the porcine MSP. The two proteins (sequence identity 51%) have a very similar 3D structure with the secondary structure elements well conserved and with most of the amino acid substitutions causing a change of charge localized to one side of the molecule. MSP is a beta-sheet-rich protein with two distinct domains. The N-terminal domain is composed of a four-stranded beta-sheet, with the strands arranged according to the Greek key-motif, and a less structured part. The C-terminal domain contains two two-stranded beta-sheets with no resemblance to known structural motifs. The two domains, connected to each other by the peptide backbone, one disulfide bond, and interactions between the N and C termini, are oriented to give the molecule a rather extended structure. This global fold differs markedly from that of a previously published structure for porcine MSP, in which the two domains have an entirely different orientation to each other. The difference probably stems from a misinterpretation of ten specific inter-domain NOEs.

Amino Acid Sequence↗
PubMed2005

Solution structure of the Ca2+-Binding EGF3-4 pair from vitamin K-dependent protein S: identification of an unusual fold in EGF3.

Vitamin K-dependent protein S is a cofactor of activated protein C, a serine protease that regulates blood coagulation. Deficiency of protein S can cause venous thrombosis. Protein S has four EGF domains in tandem; domains 2-4 bind calcium with high affinity whereas domains 1-2 mediate interaction with activated protein C. We have now solved the solution structure of the EGF3-4 fragment of protein S. The linker between the two domains is similar to what has been observed in other calcium-binding EGF domains where it provides an extended conformation. Interestingly, a disagreement between NOE and RDC data revealed a conformational heterogeneity within EGF3 due to a hinge-like motion around Glu186 in the Cys-Glu-Cys sequence, the only point in the domain where flexibility is allowed. The dominant, bent conformation of EGF3 in the pair has no precedent among calcium-binding EGF domains. It is characterized by a change in the psi angle of Glu186 from 160 degrees +/- 40 degrees , as seen in ten other EGF domains, to approximately 0 degrees +/- 15 degrees . NOESY data suggest that Tyr193, a residue not conserved in other calcium-binding EGF domains (except in the homologue Gas6), induces the unique fold of EGF3. However, SAXS data, obtained on EGF1-4 and EGF2-4, showed a dominant, extended conformation in these fragments. This may be due to a counterproductive domain-domain interaction between EGF2 and EGF4 if EGF3 is in a bent conformation. We speculate that the ability of EGF3 to adopt different conformations may be of functional significance in protein-protein interactions involving protein S.

Amino Acid Sequence↗
PubMed2005

Subtle sequence differences in a tumour-associated peptide epitope translate into major changes in antigenicity.

Antigenicity, the ability to bind to members of repertoire of diverse immune receptors, is a concept that is poorly characterised with respect to its defining parameters. To learn more about its makeup, we have investigated the ability of two peptides with highly related sequences, derived from the tumour-associated antigen mucin-1, to recruit in vitro members from a large naïve repertoire of synthetic human antibody fragments. One of the peptides represents the epitope that is immunodominant in mice. We now demonstrate that the other peptide, which differs from the first only by a very conservative aspartate-threonine to glutamate-serine change, is much less antigenic than the first peptide. This is so despite the fact that there is no observable difference in the tendency of the two peptides to adopt a structure in solution. Furthermore, the peptides differ in their immunodominant parts and the less antigenic peptide selects for antibody fragments targeting residues outside of the epitope considered to be immunodominant in mice. We conclude that subtle sequence changes greatly, affect antigenicity and immunodominance of epitopes in this important tumour-associated antigen.

Amino Acid Sequence↗
PubMed2005

NMR structure determination of proteins supplemented by quantum chemical calculations: detailed structure of the Ca2+ sites in the EGF34 fragment of protein S.

We present and test two methods to use quantum chemical calculations to improve standard protein structure refinement by molecular dynamics simulations restrained to experimental NMR data. In the first, we replace the molecular mechanics force field (employed in standard refinement to supplement experimental data) for a site of interest by quantum chemical calculations. This way, we obtain an accurate description of the site, even if a molecular-mechanics force field does not exist for this site, or if there is little experimental information about the site. Moreover, the site may change its bonding during the refinement, which often is the case for metal sites. The second method is to extract a molecular mechanics potential for the site of interest from a quantum chemical geometry optimisation and frequency calculation. We apply both methods to the two Ca2+ sites in the epidermal growth factor-like domains 3 and 4 in the vitamin K-dependent protein S and compare them to various methods to treat these sites in standard refinement. We show that both methods perform well and have their advantages and disadvantages. We also show that the glutamate Ca2+ ligand is unlikely to bind in a bidentate mode, in contrast to the crystal structure of an EGF domain of factor IX.

Binding Sites↗
PubMed2004

Stereoselective binding of levosimendan to cardiac troponin C causes Ca2+-sensitization.

The effects of the Ca(2+) sensitizer levosimendan and that of its stereoisomer dextrosimendan on the cardiac contractile apparatus were studied using skinned fibers obtained from guinea pig hearts. Levosimendan was found to be more effective than dextrosimendan in this model. The respective concentrations of levosimendan and dextrosimendan at EC(50) were 0.3 and 3 microM. In order to explain the difference in efficacy as Ca(2+) sensitizers, the binding of the two stereoisomers on cardiac troponin C was studied by nuclear magnetic resonance in the absence and presence of two peptides of cardiac troponin I. The two stereoisomers interacted with both domains of cardiac troponin C in the absence of cardiac troponin I. In the presence of cardiac troponin I-(32-79) and cardiac troponin I-(128-180), the binding of both levosimendan and dextrosimendan to the C-terminal domain of cardiac troponin C was blocked and only the binding to the N-terminal domain was observable. Differences in the overall binding behavior of the two isomers to cardiac troponin C were highlighted in order to discuss their structure to activity relation. Our data are consistent with the notion that the action of levosimendan as a Ca(2+) sensitizer and positive inotrope relates to its stereoselective binding to Ca(2+)-saturated cardiac troponin C.

Animals↗
PubMed2003

NMR solution structure of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea.

The structure of calerythrin, a prokaryotic 20 kDa calcium-binding protein has been determined by solution NMR spectroscopy. Distance, dihedral angle, J coupling, secondary chemical shift, residual dipolar coupling and radius of gyration restraints reveal four EF-hand motifs arranged in a compact globular structure. A tight turn in the middle of the amino acid sequence brings the two halves, each comprising a pair of EF-hands, close together. The structural similarity between calerythrin and the eukaryotic sarcoplasmic calcium-binding proteins is notable.

Amino Acid Sequence↗
PubMed2002

Differences in backbone dynamics of two homologous bacterial albumin-binding modules: implications for binding specificity and bacterial adaptation.

Proteins G and PAB are bacterial albumin-binding proteins expressed at the surface of group C and G streptococci and Peptostreptococcus magnus, respectively. Repeated albumin-binding domains, known as GA modules, are found in both proteins. The third GA module of protein G from the group G streptococcal strain G148 (G148-GA3) and the second GA module of protein PAB from P.magnus strain ALB8 (ALB8-GA) exhibit 59% sequence identity and both fold to form three-helix bundle structures that are very stable against thermal denaturation. ALB8-GA binds human serum albumin with higher affinity than G148-GA3, but G148-GA3 shows substantially broader albumin-binding specificity than ALB8-GA. The (15)N nuclear magnetic resonance spin relaxation measurements reported here, show that the two GA modules exhibit mobility on the picosecond-nanosecond time scale in directly corresponding regions (loops and termini). Most residues in G148-GA3 were seen to be involved in conformational exchange processes on the microsecond-millisecond time scale, whereas for ALB8-GA such motions were only identified for the beginning of helix 2 and its preceding loop. Furthermore, and more importantly, hydrogen-deuterium exchange and saturation transfer experiments reveal large differences between the two GA modules with respect to motions on the second-hour time scale. The high degree of similarity between the two GA modules with respect to sequence, structure and stability, and the observed differences in dynamics, binding affinity and binding specificity to different albumins, suggest a distinct correlation between dynamics, binding affinity and binding specificity. Finally, it is noteworthy in this context that the module G148-GA3, which has broad albumin-binding specificity, is expressed by group C and G streptococci known to infect all mammalian species, whereas P.magnus with the ALB8-GA module has been isolated only from humans.

Adaptation, Physiological↗
PubMed2002

Quaternary structure built from subunits combining NMR and small-angle x-ray scattering data.

A new principle in constructing molecular complexes from the known high-resolution domain structures joining data from NMR and small-angle x-ray scattering (SAXS) measurements is described. Structure of calmodulin in complex with trifluoperazine was built from N- and C-terminal domains oriented based on residual dipolar couplings measured by NMR in a dilute liquid crystal, and the overall shape of the complex was derived from SAXS data. The residual dipolar coupling data serves to reduce angular degrees of freedom, and the small-angle scattering data serves to confine the translational degrees of freedom. The complex built by this method was found to be consistent with the known crystal structure. The study demonstrates how approximate tertiary structures of modular proteins or quaternary structures composed of subunits can be assembled from high-resolution structures of domains or subunits using mutually complementary NMR and SAXS data.

Calmodulin↗
PubMed2002

Interaction of bovine coagulation factor X and its glutamic-acid-containing fragments with phospholipid membranes. A surface plasmon resonance study.

The interaction of blood coagulation factor X and its Gla-containing fragments with negatively charged phospholipid membranes composed of 25 mol% phosphatidylserine (PtdSer) and 75 mol% phosphatidylcholine (PtdCho) was studied by surface plasmon resonance. The binding to 100 mol% PtdCho membranes was negligible. The calcium dependence in the membrane binding was evaluated for intact bovine factor X (factor X) and the fragment containing the Gla-domain and the N-terminal EGF (epidermal growth factor)-like domain, Gla-EGFN, from factor X. Both proteins show the same calcium dependence in the membrane binding. Calcium binding is cooperative and half-maximum binding was observed at 1.5 mm and 1.4 mm, with the best fit to the experimental data with three cooperatively bound calcium ions for both the intact protein and the fragment. The dissociation constant (Kd) for binding to membranes containing 25 mol% PtdSer decreased from 4.6 microm for the isolated Gla-domain to 1 microm for the fragments Gla-EGFN and Gla-EGFNC (the Gla-domain and both EGF-like domains) fragments and to 40 nm for the entire protein as zymogen, activated enzyme or in the active-site inhibited form. Analysis of the kinetics of adsorption and desorption confirmed the equilibrium binding data.

Binding Sites↗
PubMed2001

Structure, specificity, and mode of interaction for bacterial albumin-binding modules.

We have determined the solution structure of an albumin binding domain of protein G, a surface protein of group C and G streptococci. We find that it folds into a left handed three-helix bundle similar to the albumin binding domain of protein PAB from Peptostreptococcus magnus. The two domains share 59% sequence identity, are thermally very stable, and bind to the same site on human serum albumin. The albumin binding site, the first determined for this structural motif known as the GA module, comprises residues spanning the first loop to the beginning of the third helix and includes the most conserved region of GA modules. The two GA modules have different affinities for albumin from different species, and their albumin binding patterns correspond directly to the host specificity of C/G streptococci and P. magnus, respectively. These studies of the evolution, structure, and binding properties of the GA module emphasize the power of bacterial adaptation and underline ecological and medical problems connected with the use of antibiotics.

Amino Acid Sequence↗