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Mutations as trapdoors to two competing native conformations of the Rop-dimer.

ABSTRACT: Conformational transitions play a central role in regulating protein function. Structure-based models with multiple basins have been used to understand the mechanisms governing these transitions. A model able to accommodate multiple folding basins is proposed to explore the mutational effects in the folding of the Rop-dimer (Rop). In experiments, Rop mutants show unusually strong increases in folding rates with marginal effects on stability. We investigate the possibility of two competing conformations representing a parallel (P) and the wild-type antiparallel (AP) arrangement of the monomers as possible native conformations. We observe occupation of both distinct states and characterize the transition pathways. An interesting observation from the simulations is that, for equivalent energetic bias, the transition to the P basin (non-wild-type basin) shows a lower free-energy barrier. Thus, the rapid kinetics observed in experiments appear to be the result of two competing states with different kinetic behavior, triggered upon mutation by the opening of a trapdoor arising from Rop's symmetric structure. The general concept of having competing conformations for the native state goes beyond explaining Rop's mutational behaviors and can be applied to other systems. A switch between competing native structures might be triggered by external factors to allow, for example, allosteric control or signaling.

SUBMITTER: Schug A

PROVIDER: S-EPMC2077020 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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Mutations as trapdoors to two competing native conformations of the Rop-dimer.

Schug Alexander A Whitford Paul C PC Levy Yaakov Y Onuchic José N JN

Proceedings of the National Academy of Sciences of the United States of America 20071029 45

Conformational transitions play a central role in regulating protein function. Structure-based models with multiple basins have been used to understand the mechanisms governing these transitions. A model able to accommodate multiple folding basins is proposed to explore the mutational effects in the folding of the Rop-dimer (Rop). In experiments, Rop mutants show unusually strong increases in folding rates with marginal effects on stability. We investigate the possibility of two competing confor ...[more]

PMID: 17968016

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Project description:The activation/inactivation cycle of STAT transcription factors entails their transition between different dimer conformations. Unphosphorylated STATs can dimerize in an antiparallel conformation via extended interfaces of the globular N-domains, whereas STAT activation triggers a parallel dimer conformation with mutual phosphortyrosine:SH2 domain interactions, resulting in DNA-binding and nuclear retention. However, despite the crucial role of STAT tyrosine phosphorylation in cytokine signaling, it has not been determined how this modification affects the stability and the conformational flexibility of STAT dimers. Here, we use analytical ultracentrifugation and electrophoretic mobility shift assay (EMSA) to study the association of STAT1 in solution before and after tyrosine phosphorylation. It is revealed that STAT1 formed high-affinity dimers (K(d) of approximately 50 nM) with estimated half-lives of 20-40 min irrespective of the phosphorylation status. Our results demonstrate that parallel and antiparallel conformations of STAT1 were present simultaneously, supported by mutually exclusive interfaces; and the transition between conformations occurred through affinity-driven dissociation/association reactions. Therefore, tyrosine phosphorylation was dispensable for DNA binding, but the phosphorylation enforced preformed SH2 domain-mediated dimers, thus enhancing the DNA-binding activity of STAT1 >200-fold. Moreover, upon STAT1 activation the N-domains adopted an open conformation and engaged in interdimer interactions, as demonstrated by their participation in tetramerization instead of dimerization. Yet, homotypic N-domain interactions are not conserved in the STAT family, because the N-domain dissociation constants of STAT1, STAT3, and STAT4 differed by more than three orders of magnitude. In conclusion, STAT1 constantly oscillated between different dimer conformations, whereby the abundance of the dimerization interfaces was determined by tyrosine phosphorylation.

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Mutations as trapdoors to two competing native conformations of the Rop-dimer.

Publications

Mutations as trapdoors to two competing native conformations of the Rop-dimer.

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