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Confrontation of AlphaFold models with experimental structures enlightens conformational dynamics supporting CYP102A1 functions.

ABSTRACT: Conformational dynamics plays a critical role for the function of multidomain electron transfer complexes. While crystallographic or NMR approaches allow detailed insight into structures, lower resolution methods like cryo-electron microscopy can provide more information on dynamics. In silico structure modelling using AlphaFold was recently successfully extended to the prediction of protein complexes but its capability to address large conformational changes involved in catalysis remained obscure. We used bacterial CYP102A1 monooxygenase homodimer as a test case to design a competitive modelling approach (CMA) for assessing alternate conformations of multi-domain complexes. Predictions were confronted with published crystallographic and cryo-EM data, evidencing consistencies but also permitting some reinterpretation of experimental data. Structural determinants stabilising the new type of domain connectivity evidenced in this bacterial self-sufficient monooxygenase were analysed by CMA and used for in silico retro-engineering applied to its eukaryotic bi-component counterparts.

SUBMITTER: Urban P

PROVIDER: S-EPMC9510131 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Confrontation of AlphaFold models with experimental structures enlightens conformational dynamics supporting CYP102A1 functions.

Urban Philippe P Pompon Denis D

Scientific reports 20220925 1

Conformational dynamics plays a critical role for the function of multidomain electron transfer complexes. While crystallographic or NMR approaches allow detailed insight into structures, lower resolution methods like cryo-electron microscopy can provide more information on dynamics. In silico structure modelling using AlphaFold was recently successfully extended to the prediction of protein complexes but its capability to address large conformational changes involved in catalysis remained obscu ...[more]

PMID: 36155638

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Project description:The three-dimensional structure of monomers and homodimers of CYP102A1/WT (wild-type) proteins and their A83F and A83I mutant forms was predicted using the AlphaFold2 (AF2) and AlphaFold Multimer (AFMultimer) programs, which were compared with the rate constants of hydroxylation reactions of these enzyme forms to determine the efficiency of intra- and interprotein electron transport in the CYP102A1 hydroxylase system. The electron transfer rate constants (ket), which determine the rate of indole hydroxylation by the CYP102A1 system, were calculated based on the distances (R) between donor-acceptor prosthetic groups (PG) FAD→FMN→HEME of these proteins using factor β, which describes an exponential decay from R the speed of electron transport (ET) according to the tunnelling mechanism. It was shown that the structure of monomers in the homodimer, calculated using the AlpfaFold Multimer program, is in good agreement with the experimental structures of globular domains (HEME-, FMN-, and FAD-domains) in CYP102A1/WT obtained by X-ray structural analysis, and the structure of isolated monomers predicted in AF2 does not coincide with the structure of monomers in the homodimer, although a high level of similarity in individual domains remains. The structures of monomers and homodimers of A83F and A83I mutants were also calculated, and their structures were compared with the wild-type protein. Significant differences in the structure of all isolated monomers with respect to the structures of monomers in homodimers were also found for them, and at the same time, insignificant differences were revealed for all homodimers. Comparative analysis for CYP102A1/WT between the calculated intra- and interprotein distances FAD→FMN→HEME and the rate constants of hydroxylation in these proteins showed that the distance between prosthetic groups both in the monomer and in the dimer allows the implementation of electron transfer between PGs, which is consistent with experimental literature data about kcat. For the mutant form of monomer A83I, an increase in the distance between PGs was obtained, which can restrict electron transportation compared to WT; however, for the dimer of this protein, a decrease in the distance between PGs was observed compared to the WT form, which can lead to an increase in the electron transfer rate constant and, accordingly, kcat. For the monomer and homodimer of the A83F mutant, the calculations showed an increase in the distance between the PGs compared to the WT form, which should have led to a decrease in the electron transfer rate, but at the same time, for the homodimer, the approach of the aromatic group F262 with heme can speed up transportation for this form and, accordingly, the rate of hydroxylation.

Dataset Information

Confrontation of AlphaFold models with experimental structures enlightens conformational dynamics supporting CYP102A1 functions.

Publications

Confrontation of AlphaFold models with experimental structures enlightens conformational dynamics supporting CYP102A1 functions.

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