Research ArticleInsights into the flexibility of the T3 loop and GTPase activating protein (GAP) domain of dimeric α and β tubulins from a molecular dynamics perspective
Graphical abstract
Introduction
Microtubules are cytoskeletal components of the cell that are found mainly in the cytoplasm. These cellular components of eukaryotes are critically involved in cell division, chromosomal segregation, motility and intracellular transport (Li et al., 2012; Hyams and Lloyd, 1994). Their fundamental unit - tubulin protein, is made up of α and β subunits. Each subunit is arranged in an alternate manner to form protofilaments through longitudinal interactions, thus forming intra dimer (α-β) and inter dimeric (β-α) contacts. Three distinct domains have been identified in tubulin dimers (Williams et al., 1999; Caplow et al., 1994; Löwe et al., 2001; Mickey and Howard, 1995; Correia et al., 1987). They are N-terminal domain (1–205), intermediate domain (206–381) and C-terminal domain (382–440) (Li et al., 2002; Nogales et al., 1999; Zhang et al., 1999; Amos and Löwe, 2014) (Fig. 1).
Initially, the N-terminal domain interacts with GTP in α and β subunits through the GTPase domain made up of T1-T6 loops. However, the hydrolysis of GTP to GDP is restricted to the β subunit. In particular, the T3 loop (residues 98–101) binds with the γ phosphate of GTP in both the subunits. Secondly, the intermediate domain of α and β subunits consists of GTPase Activating Protein domain (GAP)(residues 240–261) which is also present in heterotrimeric G proteins and Ras-related small GTP binding protein (Li et al., 2002; Nogales et al., 1999; Zhang et al., 1999; Amos and Löwe, 2014). Irrespective of their presence in both the subunits, only the subunit acts like GAP and assists in hydrolysis when it interacts with the GTPase domain of the β subunit (Nogales et al., 1998a, 1998b; Erickson, 1998). Basically, the GAP region is an amalgamation of two domains - viz. T7 loop (residues 240–252) and helix H8 (residues 253–261) (Nogales et al., 1999; Zhang et al., 1999; Hamel et al., 1999; Löwe and Amos, 1998; Nogales et al., 1998a, 1998b; Andreu et al., 1994). Here, the T7 loop harbours the consensus pattern GxxNxD which interacts with GTP from the adjacent domain while Glu254 from helix H8 of α subunit associates with the T3 loop of the β subunit by positioning the γ phosphate for hydrolysis in the exchangeable site (E-site) (Löwe and Amos, 1998; Nogales et al., 1998b). On the other hand, Lys254 from helix H8 of β tubulin from the Non-exchangeable site (N-site) enhances the monomer-monomer interaction in the intra-dimer interface (Fig. 2a,b) instead of GTP hydrolysis (Löwe et al., 2001; Li et al., 2002; Nogales et al., 1999; Zhang et al., 1999; Amos and Löwe, 2014). Finally, the C-terminal region interacts with the Microtubule Associated Protein (MAP) (Downing and Nogales, 1998; Nogales et al., 1998a, 1998b).
The literature survey reports that hydrolysis brings in considerable structural changes within the nucleotide binding site of the inter-dimer interface compared to the intra-dimer interface. In particular, an empty cavity is created after hydrolysis due to the loss of γ phosphate from the GTP of the inter-dimer interface of the β subunit (Löwe et al., 2001; Downing and Nogales, 1998; Nogales et al., 1998a, 1998b; Mitra and Sept, 2008; Alushin et al., 2014; Aylett et al., 2011). This cavity gets filled by the T3 loop of the β subunit and the GAP domain of the adjacent α subunit comprising of the catalytic residue Glu254 (Wang and Nogales, 2005; Li et al., 2002; Dai et al., 1994; Alushin et al., 2014; Aylett et al., 2011; Wang and Nogales, 2005; Nettles et al., 2004; Ravelli et al., 2004). The reason behind the larger internal rearrangement within the inter-dimer interface remains elusive. In the present study, we investigated the reason behind the overall flexibility of the T3 loop and GAP domain of dimeric subunits through simulation and Anisotropic Network Model (ANM) perspective. The findings of study have helped us understand the role of amino acids in the intrinsic mobility of the intra- and inter- dimeric regions.
Section snippets
Pairwise alignment and structural analysis
The protein sequences of α and β tubulin of Bos taurus was downloaded from Uniprot Database (α: P81947; β: Q6B856) (Bairoch and Apweiler, 1996). Both the sequences were considered for pairwise alignment using Clustal-Omega software (Sievers and Higgins, 2014) wherein the T3 loop and the GAP domain were explored for their amino acid conservedness. Next, their crystal structures were downloaded from the Protein Data Bank (Berman et al., 2000) (PDB ID: 4O4H) (Prota et al., 2014). This 3D structure
Sequence alignment and structural analysis
Protein sequences of α and β subunits were aligned to identify the overall amino acid conservedness within the T3 loop and GAP domain. Here we observed that, in the T3 loop of α and β subunits, the residues Ala99 and Asn101 that associate with γ phosphate interaction are conserved. In the β subunit, the residues get renumbered as Ala97 and Asn99 due to the deletion. It is the Ala99 residue of α subunit (which is flanked by Asp98 and Ala100) that positions the γ phosphate. However, it gets
Conclusion
GTP hydrolysis is a biochemical event associated with conformational changes and structural stability of microtubules. Machineries like the T3 loop and the GAP domain are integral players in this event. The presence of a glycine residue flanking the γ phosphate binding site within the T3 loop of the inter-dimer interface plays a crucial role in intrinsic domain flexibility. This movement further cascades into the GAP domain which assists in structural rearrangement. The absence of glycine
Acknowledgement
The authors would like to acknowledge the Department of Biotechnology (DBT), Government of India, sponsored Distributed Information Sub Centre (SubDIC) of Biotechnology Information System (BTIS) Network at ACTREC where the analysis of MD trajectories and docking studies was carried out. The authors thank Supercomputing facility (Sankalp) of the Computer division, BARC, for providing access for molecular dynamics simulation of structures. Lastly, the authors would also like to thank Dr. Aparna
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