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Insights into Molecular Assembly of ACCase Heteromeric Complex in Chlorella variabilis—A Homology Modelling, Docking and Molecular Dynamic Simulation Study

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Abstract

Acetyl-CoA carboxylase (ACCase), a biotin-dependent enzyme that catalyses the first committed step of fatty acid biosynthesis, is considered as a potential target for improving lipid accumulation in oleaginous feedstocks, including microalgae. ACCase is composed of three distinct conserved domains, and understanding the structural details of each catalytic domain assumes great significance to gain insights into the molecular basis of the complex formation and mechanism of biotin transport. In the absence of a crystal structure for any single heteromeric ACCase till date, here we report the first heteromeric association model of ACCase from an oleaginous green microalga, Chlorella variabilis, using a combination of homology modelling, docking and molecular dynamic simulations. The binding site of the docked biotin carboxylase (BC) and carboxyltransferase (CT) were predicted to be contiguous but distinct in biotin carboxyl carrier protein (BCCP) molecule. Simulation studies revealed considerable flexibility for the BC and CT domains in the BCCP-bound forms, thus indicating the adaptive behaviour of BCCP. Further, principal component analysis revealed that in the presence of BCCP, the BC and CT domains exhibited an open-state conformation via the outward clockwise rotation of the binding helices. These conformational changes might be responsible for binding of BCCP domain and its translocation to the respective active sites. Various rearrangements of inter-domain hydrogen bonds (H-bonds) contributed to conformational changes in the structures. H-bond interactions between the interacting residue pairs involving Glu201BCCP/Arg255BC and Asp224BCCP/Gln228CT were found to be essential for the intermolecular assembly. The present findings are consistent with previous biochemical studies.

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Acknowledgements

This work was partially funded by the Department of Biotechnology, Government of India. N.M. acknowledges the support of the Council for Scientific and Industrial Research, India for granting Senior Research Fellowship. Technical help rendered by Mr. Bikram Kumar Parida in preparation of the figures is gratefully acknowledged.

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Authors and Affiliations

  1. Academy of Scientific and Innovative Research, CSIR-Institute of Minerals and Materials Technology, Bhubaneswar, 751 013, Odisha, India

    Namrata Misra, Prasanna Kumar Panda & Barada Kanta Mishra

  2. Bioresources Engineering Department, CSIR-Institute of Minerals and Materials Technology, Bhubaneswar, 751 013, Odisha, India

    Namrata Misra & Prasanna Kumar Panda

  3. Department of Bioinformatics, Odisha University of Agriculture and Technology, Bhubaneswar, 751 003, Odisha, India

    Mahesh Chandra Patra & Sukanta Kumar Pradhan

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  1. Namrata Misra
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  2. Prasanna Kumar Panda
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Correspondence to Prasanna Kumar Panda.

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Fig. S1

Multiple sequence alignment of selected BC homologs from Chlorella variabilis (Cv, E1Z54P), Escherichia coli (Ec, P24182), Staphylococcus aureus (Sa, Q99TW7), Cyanidioschyzon merolae (Cm, CMS299C), Arabidopsis thaliana (At, O04983), Brassica napus (Bn, D9I767), Ricinus communis (Rc, B9S1E2), Jatropha curcas (Jc, F2WMV4), Glycine max (Gm, O23960), Arachis hypogaea (Ah, E6Y6R4), Elaeis guineinsis (Eg, D2CFM8), and Volvox carteri (Vc, D8UF54). ‘▼’ indicates amino acids lying within 4 Å of bound ATP. ‘*’ denotes conserved amino acids in all sequences. The residues of the P-loop region are boxed. The color code for the residues and the conservation of pattern follows the Clustal program (JPEG 79 kb)

High resolution image (TIFF 371 kb)

Fig. S2

Multiple sequence alignment of the biotinyl domain of BCCP homologs from Chlorella variabilis (Cv, E1Z723), Volvox carteri (Vc, D8U256), Chlamydomonas reinhardtii (Cr, A8JDA7), Anabaena variabilis (Av, Q3WAQ4), Ricinus communis (Rc, B9RM56), Jatropha curcas (Jc, F2WMV5), Glycine max (Gm, Q42783), Arabidopsis thaliana (At, F4KE21), Brassica napus (Bn, G4WXD7), Arachis hypogaea (Ah, E6Y6R2), Escherichia coli (Ec, P0ABD8), and Cyanidioschyzon merolae (Cm, CMV134C). ‘▼’ indicates the core hydrophobic residues. ‘●’ indicates residues involved in binding to BC domain. ‘▀’ indicates residues involved in binding to CT domain. ‘*’ denotes conserved amino acids in all sequences. The residues of the thumb regions are boxed. The color code for the residues and the conservation of pattern follows the Clustal program (JPEG 12 kb)

High resolution image (TIFF 436 kb)

Fig. S3

Multiple sequence alignment of the Zinc domain present in β subunit of CT homologs from Chlorella variabilis (Cv, F2YGI4), Chlamydomonas reinhardtii (Cr, A8JHU1), Volvox carteri (Vc, D8U455), Anabaena variabilis (Av, Q3MGS5), Cyanidioschyzon merolae (Cm, CMV207C), Arabidopsis thaliana (At, P56765), Brassica napus (Bn, G4XGU6), Glycine max (Gm, P49158), Ricinus communis (Rc, G1D767), and Escherichia coli (Ec, D3QL09). The conserved Cysteine residues of ‘Zinc ribbon motif’ is indicated by ‘*’. The color code for the residues and the conservation of pattern follows the Clustal program (JPEG 4 kb)

High resolution image (TIFF 128 kb)

Fig. S4

ERRAT plot for the models. a BC domain. b BCCP domain. c α subunit of CT domain. d β subunit of CT domain. The ‘overall quality factor’ indicates the percentage of the protein for which the calculated error value falls below the 95 % rejection limit, respectively. (JPEG 30 kb)

High resolution image (TIFF 399 kb)

Fig. S5

PROSA plot showing the location of the Z-score for the models. a BC domain. b BCCP domain. c α subunit of CT domain. d β subunit of CT domain. The calculated Z-score for all the developed models are in the range of experimentally determined protein structure present in PDB (JPEG 18 kb)

High resolution image (TIFF 168 kb)

Fig. S6

PROSA energy plot for the models. a BC domain. b BCCP domain. c α subunit of CT domain. d β subunit of CT domain. The plot signifies the local model quality by plotting energies as a function of amino acid sequence where positive values indicate erroneous parts. Overall, the residue energies of all the models are largely negative (JPEG 17 kb)

High resolution image (TIFF 209 kb)

Fig. S7

Ramachandran plot for the models. a BC domain. b BCCP domain. c α subunit of CT domain. d β subunit of CT domain. The most favoured, additional allowed, generously allowed and disallowed regions are represented in red, deep yellow, light yellow, and white, respectively (JPEG 48 kb)

High resolution image (TIFF 400 kb)

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Misra, N., Panda, P.K., Patra, M.C. et al. Insights into Molecular Assembly of ACCase Heteromeric Complex in Chlorella variabilis—A Homology Modelling, Docking and Molecular Dynamic Simulation Study. Appl Biochem Biotechnol 170, 1437–1457 (2013). https://doi.org/10.1007/s12010-013-0277-0

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