Axel Wollmer

Targeted molecular dynamics simulation of conformational change: application to the T↔R transition in insulin

Abstract A novel method to calculate transition pathways between two known protein conformations is presented. It is based on a molecular dynamics simulation starting from one conformational state as initial structure and using the other for a directing constraint. The method is exemplified with the T ↔ R transition of insulin. The most striking difference between these conformational states is that in T the 8 N-terminal residues of the B chain are arranged as an extended strand whereas in R they are forming a helix. Both the transition from T to R and from R to T were simulated. The method proves capable of finding a continuous pathway for each direction which are moderately different. The refolding processes are illustrated by a series of transient structures and pairs of O, ψ angles selected from the time course of the simulations. In the T → R direction the helix is formed in the →last third of the transition, while in the R → T direction it is preserved during more than half of the simulation period....

Epidermal growth factor binding induces a conformational change in the external domain of its receptor

To study the properties of the extracellular epidermal growth factor (EGF) binding domain of the human EGF receptor, we have infected insect cells with a suitably engineered baculovirus vector containing the cDNA encoding the entire ectodomain of the parent molecule. This resulted in a correctly folded, stable, 110 kd protein which possessed an EGF binding affinity of 200 nM. The protein was routinely purified in milligram amounts from 1 litre insect cell cultures using a series of three standard chromatographic steps. The properties of the ectodomain were studied before and after the addition of different EGF ligands, using both circular dichroism and fluorescence spectroscopic techniques. A secondary structural analysis of the far UV CD spectrum of the ectodomain indicated significant proportions of alpha‐helix and beta‐sheet in agreement with a published model of the EGF receptor. The ligand additions to the receptor showed differences in both the near‐ and far‐UV CD spectra, and were similar for each ligand used, suggesting similar conformational differences between uncomplexed and complexed receptor. Steady‐state fluorescence measurements indicated that the tryptophan residues present in the ectodomain are buried and that the solvent‐accessible tryptophans in the ligands become buried on binding the receptor. The rotational correlation times measured by fluorescence anisotropy decay for the receptor‐ligand complexes were decreased from 6 to 2.5 ns in each case. This may indicate a perturbation of the tryptophan environment of the receptor on ligand binding. Ultracentrifugation studies showed that no aggregation occurred on ligand addition, so this could not explain the observed differences from CD or fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-function analysis of human interleukin-6 : evidence for the involvement of the carboxy-terminus in function

C‐terminally deleted analogs of human interleukin‐6 (IL‐6) have been constructed at the cDNA level, and after cell‐free transcription and translation their biological activity was analyzed. Removal of only 4 amino acids resulted in complete loss of biological activity as determined by the B9 cell proliferation assay. Secondary structure prediction of human IL‐6 resulted in 58% helix, 14% β‐structure, and 28% turn and coil (average of 3 independent methods). The circular dichroism of recombinant human IL‐6 was measured in the near and far UV. Evaluation of the latter in terms of secondary structures gave 67% helix, 15% β‐structure, and 18% turn and coil.

The family of the IL-6-type cytokines: specificity and promiscuity of the receptor complexes.

The cytokines IL‐6, LIF, CNTF, OSM, IL‐11, and CT‐1 have been grouped into the family of IL‐6‐type cytokines, since they all require gp130 for signal transduction. Interestingly, gp130 binds directly to OSM, whereas complex formation with the other cytokines depends on additional receptor subunits. Only limited structural information on these cytokines and their receptors is available. X‐ray structures have been solved for the cytokines LIF and CNTF, whose up‐up‐down‐down four‐helix bundle is common to all of these cytokines, and for the receptors of hGH and prolactin, which contain two domains with a fibronectin III‐like fold. Since cocrystallization and x‐ray analysis of the up to four different proteins forming the receptor complexes of the IL‐6‐type cytokines is unlikely to be achieved in the near future, model building based on the existing structural information is the only approach for the time being. Here we present model structures of the complexes of human and murine IL‐6 with their receptors. Their validity can be deduced from the fact that published mutagenesis data and the different receptor specificity of human and murine IL‐6 can be understood. It is now possible to predict the relative positions and contacts for all molecules in their respective complexes. Such information can be used for the rational design of cytokine and receptor antagonists, which may have a valuable therapeutic perspective. Proteins 27:96–109

Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes.

Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3‐kinase, a closely related p85 beta protein, and a recombinant SH2 domain‐containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase‐insert region of the platelet‐derived growth factor beta‐receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain‐containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain‐containing proteins is discussed.

Adaptation of plasminogen activator sequences to known protease structures

The sequences of urokinase (UK) and tissue‐type plasminogen activator (TPA) were aligned with those of chymotrypsin, trypsin, and elastase according to their ‘structurally conserved regions’. In spite of its trypsin‐like specificity UK was model‐built on the basis of the chymotrypsin structure because of a corresponding disulfide pattern. The extra disulfide bond falls to cysteines 50 and 111d. Insertions can easily be accommodated at the surface. As they occur similarly in both, UK and TPA, a role in plasminogen recognition may be possible. Of the functional positions known to be involved in substrate or inhibitor binding, Asp 97, Lys 143 and Arg 217 (Leu in TPA) may contribute to plasminogen activating specificity. PTI binding may in part be impaired by structural differences at the edge of the binding pocket.

Calculations of the CD spectrum of bovine pancreatic ribonuclease

CD spectra of bovine pancreatic ribonuclease A (RNase A) and its subtilisin-modified from (RNase S) have been calculated, based upon high-resolution structures from x-ray diffraction. All known transitions in the peptide and side-chain groups, especially the aromatic and disulfide groups, have been included. Calculations have been performed with both the matrix method and with first-order perturbation theory. A newly developed method for treating the electrostatic interactions among transition charge densities and between static charge distributions and transition charge densities is used. The effects of local electrostatic fields upon the group transition energies are included for all transitions. Rotational strengths generated by the matrix method were combined with Gaussian band shapes to generate theoretical CD spectra. The calculated spectra reproduce the signs and approximate magnitudes of the near-uv CD bands of both RNase A and S. Agreement is most satisfactory for the negative 275 nm band, dominated by tyrosine contributions. In agreement with two previous studies by other workers, coupling between Tyr 73 and Tyr 115 is the single most important factor in this band. The positive band observed near 240 nm is dominated by disulfide contributions, according to our results. The far-uv CD spectrum is poorly reproduced by the calculations. The observed 208 nm band, characteristic of alpha-helices, is absent from the calculated spectrum, probably because the helices in RNase are short. A strong positive couplet centered near 190 nm is predicted but not observed. Possible reasons for these incorrect predictions of the current theoretical model in the far-uv are discussed.

A COMPARISON OF THE STRUCTURE AND DYNAMICS OF AVIAN PANCREATIC-POLYPEPTIDE HORMONE IN SOLUTION AND IN THE CRYSTAL

A molecular dynamics simulation was carried out with avian pancreatic polypeptide hormone (aPP) as an isolated monomer explicitly including the solvent (MDS). The simulation and the resulting mean structure are compared with the results of a corresponding crystal simulation (MDC) with 4 aPP molecules plus interstitial water in a periodic boundary unit cell and with the X-ray structure (van Gunsteren, Haneef et al., manuscript in preparation). Comparison is based on the time span 5 to 15 ps and considering cartesian coordinates, dihedral angles, H-bond length, and accessible surface area. While in the MDC simulation equilibration is fast and complete, it does occur in MDS for most but not all parts of the molecule; the turn region starts moving away from the X-ray structure after 9 ps.Only minor differences result when dimerforming side chains, e.g. tyrosines 7 and 21, are exposed to solvent. The largest rms fluctuations are encountered in exposed polar side chains of Asp 11, Glu 15, Arg 19, and Arg 33, but also in the hydrophobic core residue Phe 20, the only phenylalanine residue present. The latter undergoes an abrupt reorientation suitable for verification by NMR spectroscopy, which is possibly related to the motion of the turn region. The main-chain dihedral angles of the α-helix are shifted from values generally found in crystal structures towards those of the ideal Pauling helix. There is concomitant H-bond elongation. The effects are most pronounced and consistent in the MDS simulation.

A monomeric insulin from the porcupine (Hystrix cristata), an Old World hystricomorph

The insulins of New World hystricomorph rodents exhibit many novel amino acid changes in primary structure when compared with other mammalian insulins1–3. These changes give rise to unusual properties (low potency, failure to self-associate4–6) not shared by other naturally-occurring insulins. We report here on the primary structure, zinc-binding properties and circular dichroism (CD) of porcupine insulin (Hystrix cristata), the first Old World hystricomorph insulin to be investigated, and discuss the changes in primary structure of the hormone in relation to its properties. Residue B22 is strongly implicated as being responsible for the unusual properties of porcupine insulin.

Characterization and Binding Specificity of the Monomeric STAT3-SH2 Domain

Signal transducers and activators of transcription (STATs) are important mediators of cytokine signal transduction. STAT factors are recruited to phosphotyrosine-containing motifs of activated receptor chains via their SH2 domains. The subsequent tyrosine phosphorylation of the STATs leads to their dissociation from the receptor, dimerization, and translocation to the nucleus. Here we describe the expression, purification, and refolding of the STAT3-SH2 domain. Proper folding of the isolated protein was proven by circular dichroism and fluorescence spectroscopy. The STAT3-SH2 domain undergoes a conformational change upon dimerization. Using an enzyme-linked immunosorbent assay we demonstrate that the monomeric domain binds to specific phosphotyrosine peptides. The specificity of binding to phosphotyrosine peptides was assayed with the tyrosine motif encompassing Tyr705 of STAT3 and with all tyrosine motifs present in the cytoplasmic tail of the signal transducer gp130.