ABSTRACT
The hormonal precursor pro-ocytocin-neurophysin is activated by selective cleavage at Arg2-Ala13, producing mature ocytocin and neurophysin. To understand the cleavage mechanism better, and in particular the recognition of the cleavage site, it is necessary to characterize the three-dimensional structure of the precursor molecule. Here we combine a variety of experimental data with molecular modeling and dynamics calculations to derive possible precursor conformations. In the models obtained, the N-terminus of the precursor, corresponding to the ocytocin segment, is hydrogen bonded in a pocket of the neurophysin moiety in a similar manner to a crystallographically obtained non-covalent complex between the two molecules. The calculations suggest that although the ocytocin segment is relatively flexible, it adopts a stable, broad loop structure in the vicinity of the cleavage region, which may constitute the structural element recognized by the cleaving enzyme. The calculations also suggest a possible widening of the distance between the two neurophysin domains in the precursor relative to that in the non-covalent neurophysin-ocytocin complex.
Subject(s)
Models, Molecular , Neurophysins/chemistry , Oxytocin/chemistry , Protein Conformation , Protein Precursors/chemistry , Amino Acid Sequence , Animals , Binding Sites , Cattle , Computer Simulation , Crystallization , Disulfides/chemistry , Disulfides/metabolism , Hydrogen Bonding , Molecular Sequence Data , Neurophysins/metabolism , Nuclear Magnetic Resonance, Biomolecular , Oxytocin/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Folding , Protein Precursors/metabolism , ThermodynamicsABSTRACT
In this paper, we explore the possibility of using experimental observations in the Monte Carlo chain growth method that we have previously developed. In this method, the macromolecule (peptide, protein, nuclei acid, etc.) is grown atom-by-atom (or residue-by-residue, etc.) and partial chains are replicated according to their Boltzmann weights. Once the molecule completed, we are left with a Boltzmann-distributed ensemble of configurations. For long molecules, an efficient sampling of the (extremely large) phase space is difficult for obvious reasons (existence of many local minima, limited computer memory, etc.). In the case in which one is mainly interested in the low energy conformations, we have incorporated in the growth scheme experimental observations taken from the Protein Data Banks. More precisely, we have considered the case of twenty-L-alanine and we have used the (experimental) Ramachandran's plot for this residue. The biased growth procedure goes as follows: (a) each time one adds along the main backbone chain, either a carbon atom belonging to a carbonyl group, or a nitrogen atom, its dihedral angle (theta) or (psi) is drawn with a probability law that reflects the experimental Ramachandran (theta, psi) plot; (b) the bias introduced in this way is canceled through an extra term in the energy (replication energy = true energy + bias energy); (c) the configurations, generated at T = 1000 K, are then energy minimized.(ABSTRACT TRUNCATED AT 250 WORDS)
