Søren M. Kristensen

Solution structure of the C-terminal SH2 domain of the p85 alpha regulatory subunit of phosphoinositide 3-kinase.

Heterodimeric class IA phosphoinositide 3-kinase (PI 3-kinase) plays a crucial role in a variety of cellular signalling events downstream of a number of cell-surface receptor tyrosine kinases. Activation of the enzyme is effected in part by the binding of two Src homology-2 domains (SH2) of the 85 kDa regulatory subunit to specific phosphotyrosine-containing peptide motifs within activated cytoplasmic receptor domains. The solution structure of the uncomplexed C-terminal SH2 (C-SH2) domain of the p85 alpha subunit of PI 3-kinase has been determined by means of multinuclear, double and triple-resonance NMR experiments and restrained molecular-dynamics simulated-annealing calculations. The solution structure clearly indicates that the uncomplexed C-SH2 domain conforms to the consensus polypeptide fold exhibited by other SH2 domains, with an additional short helical element at the N terminus. In particular, the C-SH2 structure is very similar to both the p85 alpha N-terminal SH2 domain (N-SH2) and the Src SH2 domain with a root mean square difference (rmsd) for 44 C alpha atoms of 1.09 and 0.89 A, respectively. The canonical BC, EF and BG loops are less well-defined by the experimental restraints and show greater variability in the ensemble of C-SH2 conformers. The lower level of definition in these regions may reflect the presence of conformational disorder, an interpretation supported by the absence or broadening of backbone and side-chain NMR resonances for some of these residues. NMR experiments were performed, where C-SH2 was titrated with phosphotyrosine-containing peptides corresponding to p85 alpha recognition sites in the cytoplasmic domain of the platelet-derived growth-factor receptor. The ligand-induced chemical-shift perturbations indicate the amino-acid residues in C-SH2 involved in peptide recognition follow the pattern predicted from homologous complexes. A series of C-SH2 mutants was generated and tested for phosphotyrosine peptide binding by surface plasmon resonance. Mutation of the invariant Arg36 (beta B5) to Met completely abolishes phosphopeptide binding. Mutation of each of Ser38, Ser39 or Lys40 in the BC loop to Ala reduces the affinity of C-SH2 for a cognate phosphopeptide, as does mutation of His93 (BG5) to Asn. These effects are consistent with the involvement of the BC loop and BG loops regions in ligation of phosphopeptide ligands. Mutation of Cys57 (beta D5) in C-SH2 to Ile, the corresponding residue type in the p85 alpha N-SH2 domain, results in a change in peptide binding selectivity of C-SH2 towards that demonstrated by p85 alpha N-SH2. This pattern of p85 alpha phosphopeptide binding specificity is interpreted in terms of a model of the p85 alpha/PDGF-receptor interaction.

Enhanced Stability of a Protein with Increasing Temperature

The unusual stability of a structured but locally flexible protein, human growth hormone (hGH) at pH 2.7, was investigated using the temperature dependence of the nanosecond-picosecond dynamics of the backbone amide groups obtained from (15)N NMR relaxation data. It is found that the flexibility of the backbone of the helices decreases with temperature in the range from 24 °C to ∼40 °C, corresponding to an increasing stability. A concomitant increase with temperature of the electrostatic interactions between charged residues forming an interhelical network of salt bridges at the center of the four-helix core suggests that these interactions give rise to the decreasing flexibility and increasing stability of the protein. However, numerous hydrophobic interactions in the interior of the four-helix core may also contribute. Above ∼40 °C, where the thermal energy overcomes the electrostatic and hydrophobic interactions, a substantial increase in the flexibility of the helix backbones results in a highly positive contribution from the local conformational heat capacity, C(p, conf), of the helix backbones to the total heat capacity, C(p), of the protein. This reduces the change in heat capacity upon unfolding, ΔC(p), increases the change in the Gibbs free energy, ΔG(unfold), and stabilizes the protein at high temperatures. A similar decrease in flexibility is found near other salt bridges in hGH and in Calmodulin and may be of general importance for the thermostability of proteins and, in particular, of the salt bridge intensive thermophilic proteins.

Proton nuclear magnetic resonance study of the B9(Asp) mutant of human insulin: Sequential assignment and secondary structure

The sequence-specific 1H nuclear magnetic resonance (n.m.r.) assignment of 49 of the 51 amino acid residues of human B9(Asp) insulin in water at low pH is reported. Spin systems were identified using a series of two-dimensional n.m.r. techniques. For the majority of the amino acid residues with unique spin systems, particularly Ala, Thr, Val, Leu, Ile and Lys, the complete spin systems were identified. Sequence-specific assignments were obtained from sequential nuclear Overhauser enhancement (NOE) connectivities. The results indicate that the solution structure of the mutant closely resembles the crystal structure of native insulin. Thus, the NOE data reveal three helical domains all consistent with the secondary structure of the native human 2Zn insulin in the crystal phase. Numerous slowly exchanging amide protons support these structural elements, and indicate a relatively stable structure of the protein. A corresponding resemblance of the tertiary structures in the two phases is also suggested by slowly exchanging amide protons, and by the extreme chemical shift values observed for the beta-protons of B15(Leu) that agree with a close contact between this residue and the aromatic rings of B24(Phe) and B26(Tyr), as found in the crystal structure of the 2Zn insulin. Finally, there are clear indications that the B9(Asp) insulin mutant exists primarily as a dimer under the given conditions.

Short strong hydrogen bonds in proteins: a case study of rhamnogalacturonan acetylesterase

The short hydrogen bonds in rhamnogalacturonan acetylesterase have been investigated by structure determination of an active-site mutant, 1H NMR spectra and computational methods. Comparisons are made to database statistics. A very short carboxylic acid carboxylate hydrogen bond, buried in the protein, could explain the low-field (18 p.p.m.) 1H NMR signal.

Weak self-association of human growth hormone investigated by nitrogen-15 NMR relaxation.

The self‐association of human growth hormone (hGH) was investigated using 15N NMR relaxation. The investigation relies on the 15N R1 and R2 relaxation rates and the heteronuclear {1H}‐15N NOEs of the backbone amide groups at multiple protein concentrations. It is shown that the rotational correlation time of hGH in solution depends strongly on its concentration, indicating a significant degree of self‐association. The self‐association is reversible and the monomers in the aggregates are noncovalently linked. Extrapolation of the relaxation data to zero concentration predicts a correlation time of 13.4 ns and a rotational diffusion anisotropy of 1.26 for monomeric hGH, in agreement with the rotational diffusion properties estimated by hydrodynamic calculations. Moreover, the extrapolation allows characterization of the backbone dynamics of monomeric hGH without interference from self‐association phenomena, and it is found that hGH is considerably more flexible than originally thought. A concerted least‐squares analysis of the 15N relaxations and their concentration dependence reveals that the self‐association goes beyond a simple monomer‐dimer equilibrium, and that tetramers or other multimeric states co‐exist in fast exchange with the monomeric and dimeric hGH at sub‐millimolar concentrations. Small changes in the 1H and 15N amide chemical shifts suggest that a region around the C‐terminus is involved in the oligomer formation. Proteins 2008.

Solution structure of the C-terminal SH2 domain of the p85α regulatory subunit of phosphoinositide 3-kinase

Heterodimeric class IA phosphoinositide 3-kinase (PI 3-kinase) plays a crucial role in a variety of cellular signalling events downstream of a number of cell-surface receptor tyrosine kinases. Activation of the enzyme is effected in part by the binding of two Src homology-2 domains (SH2) of the 85 kDa regulatory subunit to specific phosphotyrosine-containing peptide motifs within activated cytoplasmic receptor domains. The solution structure of the uncomplexed C-terminal SH2 (C-SH2) domain of the p85 alpha subunit of PI 3-kinase has been determined by means of multinuclear, double and triple-resonance NMR experiments and restrained molecular-dynamics simulated-annealing calculations. The solution structure clearly indicates that the uncomplexed C-SH2 domain conforms to the consensus polypeptide fold exhibited by other SH2 domains, with an additional short helical element at the N terminus. In particular, the C-SH2 structure is very similar to both the p85 alpha N-terminal SH2 domain (N-SH2) and the Src SH2 domain with a root mean square difference (rmsd) for 44 C alpha atoms of 1.09 and 0.89 A, respectively. The canonical BC, EF and BG loops are less well-defined by the experimental restraints and show greater variability in the ensemble of C-SH2 conformers. The lower level of definition in these regions may reflect the presence of conformational disorder, an interpretation supported by the absence or broadening of backbone and side-chain NMR resonances for some of these residues. NMR experiments were performed, where C-SH2 was titrated with phosphotyrosine-containing peptides corresponding to p85 alpha recognition sites in the cytoplasmic domain of the platelet-derived growth-factor receptor. The ligand-induced chemical-shift perturbations indicate the amino-acid residues in C-SH2 involved in peptide recognition follow the pattern predicted from homologous complexes. A series of C-SH2 mutants was generated and tested for phosphotyrosine peptide binding by surface plasmon resonance. Mutation of the invariant Arg36 (beta B5) to Met completely abolishes phosphopeptide binding. Mutation of each of Ser38, Ser39 or Lys40 in the BC loop to Ala reduces the affinity of C-SH2 for a cognate phosphopeptide, as does mutation of His93 (BG5) to Asn. These effects are consistent with the involvement of the BC loop and BG loops regions in ligation of phosphopeptide ligands. Mutation of Cys57 (beta D5) in C-SH2 to Ile, the corresponding residue type in the p85 alpha N-SH2 domain, results in a change in peptide binding selectivity of C-SH2 towards that demonstrated by p85 alpha N-SH2. This pattern of p85 alpha phosphopeptide binding specificity is interpreted in terms of a model of the p85 alpha/PDGF-receptor interaction.

Elucidation of the origin of multiple conformations of the human α3-chain type VI collagen C-terminal Kunitz domain: The reorientation of the Trp21 ring

SummaryThe human α3-chain type VI collagen C-terminal Kunitz domain fragment (α3(VI)) has been studied by two-dimensional 1H−1H and 1H−13C NMR spectroscopy at 303 K. It is shown that the secondary structure of the protein is strikingly similar to that of BPTI, and that a number of unusual Hα chemical shifts, which are highly conserved in Kunitz-domain proteins, are also observed for α3(VI). Further-more a series of exchange cross peaks observed in 1H−1H spectra shows that a large number of protons in the central β-sheet exist in two different chemical environments, corresponding to two unequally populated conformations that are slowly exchanging on the NMR time scale. Several protons, including Ser47(53) Hα, Arg32(38) Hγ2, and Gln48(54) Hβ2, all located in the vicinity of the Trp21(27) ring in the crystal structure of α3(VI) [Arnoux, B. et al. (1995) J. Mol. Biol., 246, 609–617], have very different chemical shifts in the two conformations, the most affected being Gln48(54) Hβ2 (Δδ=1.53 ppm), which is placed directly above the Trp21(27) ring in the crystal structure of α3(VI). It is concluded that the origin of the multiple conformations of the central β-sheet is a reorientation of the Trp21(27) ring. From the intensities of corresponding signals in the two conformations, the population of the minor conformation was found to be 6.4±0.2% of that of the major conformation, while a rate constant kM=1.01±0.05 s-1 for the major to minor interconversion was obtained from a series of NOESY spectra with different mixing times. In addition, it is shown that Cys14(20)-Cys38(44) disulfide bond isomerization, previously observed in BPTI [Otting, G. et al. (1993) Biochemistry, 32, 3570–3582], is also likely to occur in α3(VI).

Assignment of the backbone carbonyl resonances in 15N-labelled proteins with 13C at natural abundance by a 2D triple-resonance correlation technique

SummaryA 2D NMR experiment for assignment of backbone carbon resonances in small and medium-sized 15N-labelled proteins with 13C at natural abundance is presented. The experiment is a two-dimensional variant of the HNCO triple-resonance experiment and is demonstrated by application to a 6 kDa protein at relatively low concentration (2 mM) and temperature (30°C). The experiment is particularly suitable for assignment of carbonyl resonances.

Solution structure of the C-terminal SH2 domain of the p85α regulatory subunit of phosphoinositide 3-kinase 1 1Edited by P. E. Wright

Heterodimeric class IA phosphoinositide 3-kinase (PI 3-kinase) plays a crucial role in a variety of cellular signalling events downstream of a number of cell-surface receptor tyrosine kinases. Activation of the enzyme is effected in part by the binding of two Src homology-2 domains (SH2) of the 85 kDa regulatory subunit to specific phosphotyrosine-containing peptide motifs within activated cytoplasmic receptor domains. The solution structure of the uncomplexed C-terminal SH2 (C-SH2) domain of the p85 alpha subunit of PI 3-kinase has been determined by means of multinuclear, double and triple-resonance NMR experiments and restrained molecular-dynamics simulated-annealing calculations. The solution structure clearly indicates that the uncomplexed C-SH2 domain conforms to the consensus polypeptide fold exhibited by other SH2 domains, with an additional short helical element at the N terminus. In particular, the C-SH2 structure is very similar to both the p85 alpha N-terminal SH2 domain (N-SH2) and the Src SH2 domain with a root mean square difference (rmsd) for 44 C alpha atoms of 1.09 and 0.89 A, respectively. The canonical BC, EF and BG loops are less well-defined by the experimental restraints and show greater variability in the ensemble of C-SH2 conformers. The lower level of definition in these regions may reflect the presence of conformational disorder, an interpretation supported by the absence or broadening of backbone and side-chain NMR resonances for some of these residues. NMR experiments were performed, where C-SH2 was titrated with phosphotyrosine-containing peptides corresponding to p85 alpha recognition sites in the cytoplasmic domain of the platelet-derived growth-factor receptor. The ligand-induced chemical-shift perturbations indicate the amino-acid residues in C-SH2 involved in peptide recognition follow the pattern predicted from homologous complexes. A series of C-SH2 mutants was generated and tested for phosphotyrosine peptide binding by surface plasmon resonance. Mutation of the invariant Arg36 (beta B5) to Met completely abolishes phosphopeptide binding. Mutation of each of Ser38, Ser39 or Lys40 in the BC loop to Ala reduces the affinity of C-SH2 for a cognate phosphopeptide, as does mutation of His93 (BG5) to Asn. These effects are consistent with the involvement of the BC loop and BG loops regions in ligation of phosphopeptide ligands. Mutation of Cys57 (beta D5) in C-SH2 to Ile, the corresponding residue type in the p85 alpha N-SH2 domain, results in a change in peptide binding selectivity of C-SH2 towards that demonstrated by p85 alpha N-SH2. This pattern of p85 alpha phosphopeptide binding specificity is interpreted in terms of a model of the p85 alpha/PDGF-receptor interaction.