The molecular chaperone heat-shock protein 90 (HSP90) functions as a homodimer. Each HSP90 protomer contains three flexibly linked regions, the N-terminal ATP-binding domain (NTD), the middle domain, and the C-terminal dimerization domain (Prodromou C et al. 1997; Pearl LH and Prodromou C 2006). HSP90 dimer is rather a dynamic molecule and ATP binding and hydrolysis are associated with conformational changes (Obermann WM et al. 1998; Krukenberg KA et al. 2011; Li J & Buchner J 2013; Prodromou C 2012). The structures of the isolated yeast and human N-terminal domain (NTD) of HSP90 bound to ATP, ADP and adenylylimidodiphosphate (AMP-PNP, a non-hydrolysable analogue of ATP) suggest that nucleotides bind deep in the cleft of NTD in open apo state of HSP90 (Prodromou C et al.1997; Meyer P et al. 2003, 2004; Colombo G et al. 2008; Li J et al. 2012). The structural studies of NTD of human HSP90 with antitumor agent geldanamycin (that acts as an ADP/ATP mimetic) support the polar interactions in the binding pocket described for yeast Hsp90 and ADP or ATP (Stebbins CE et al. 1997; Prodromou C et al.1997; Grenert JP et al. 1997). Once ATP is bound it helps to stabilize the closed ATP lid state, in which the gamma-phosphate of ATP provides a hydrogen bonding that promotes a stable association of the ATP lid with NTD. The association of ATP or AMP-PNP with NTD then stimulates structural changes in NTD. NMR analysis of human full-length HSP90 protein with and without ATP confirmed that ATP binding led to conformational changes in NTD (Karagöz GE et al. 2010). No structural changes were observed in the middle and C-terminal domains (Karagöz GE et al. 2010). However, other studies suggest that ATP-dependent conformational changes occur both in NTD and in the middle domain of HSP90 (Ali MM et al. 2006; Prodromou C et al. 2000; Chadli A et al. 2000; Meyer P et al. 2003). The changes are likely to involve movements of the ATP lid segment within each N-terminal domain that locates over the bound ATP (Ali MM et al. 2006; Prodromou C et al. 2000; Chadli A et al. 2000). The movement of the lids exposes surface residues that are subsequently involved in transient dimerization of the N-terminal domains of HSP90 (Ali MM et al. 2006; Prodromou C et al. 2000; Chadli A et al. 2000). The subsequent conformational changes upon ATP binding are regulated by co-chaperone activities. For example, arrangement of the STIP1 domains in the complex seems to prevent the NTDs dimerization of HSP90 monomers and total closure of the HSP90 dimer that is required for an efficient HSP90-mediated ATP hydrolysis (Southworth DR & Agard DA 2011; Alvira S et al. 2014). Thus, ATP binding coupled to co-chaperone-mediated loading of client protein to HSP90 complex regulates ATPase activity of HSP90.