A novel ATP-dependent conformation in p97 N–D1 fragment revealed by crystal structures of disease-related mutants

Abstract

Mutations in p97, a major cytosolic AAA (ATPases associated with a variety of cellular activities) chaperone, cause inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia (IBMPFD). IBMPFD mutants have single amino-acid substitutions at the interface between the N-terminal domain (N-domain) and the adjacent AAA domain (D1), resulting in a reduced affinity for ADP. The structures of p97 N-D1 fragments bearing IBMPFD mutations adopt an atypical N-domain conformation in the presence of Mg(2+).ATPgammaS, which is reversible by ADP, showing for the first time the nucleotide-dependent conformational change of the N-domain. The transition from the ADP- to the ATPgammaS-bound state is accompanied by a loop-to-helix conversion in the N-D1 linker and by an apparent re-ordering in the N-terminal region of p97. X-ray scattering experiments suggest that wild-type p97 subunits undergo a similar nucleotide-dependent N-domain conformational change. We propose that IBMPFD mutations alter the timing of the transition between nucleotide states by destabilizing the ADP-bound form and consequently interfere with the interactions between the N-domains and their substrates.

Control of p97 function by cofactor binding

“…Coupled to this large rearrangement is a loop-to-helix transition in the linker connecting the N and D1 domains. While currently no crystal structure of the wild-type N-D1 fragment in the presence of ATPcS is available, SAXS studies suggest a similar nucleotide-dependent motion [29].…”

“…For example, the maximal difference in the N-D1-D2 angle between different states is 11°, but also for these intra-protomer domain rearrangements it is unclear whether crystal contacts prevented more significant changes. Crystal structures of N-D1 fragments of the wild-type and IBMPFD-associated mutant variants, however, did reveal dramatic conformational changes in the position of the N domain [29]. It undergoes a displacement by $12 Å and a rotation of more than 90°from its position in plane with the D1 ring (''down" conformation) as observed in all full-length crystal structures as well as in the ADP state of wild-type and IBMPFD mutant N-D1 fragments, to an out of plane (''up") conformation present in N-D1 structures of IBMPFD mutants in the presence of ATPcS (Fig.…”

“…Due to an altered inter-subunit communication in IBMPFD mutants the D1 domains fail to regulate their respective nucleotide-binding states as evidenced by a lower amount of pre-bound ADP and weaker affinity for ADP, resulting in a better accessibility for ATP in comparison to the wild-type protein [29,30]. The altered conformational equilibrium of the N domain in p97 disease mutants is accompanied by elevated ATPase activities in vitro [43,115,116] and altered interactions with some but not all cofactors in cells, which has been proposed to be key to the pathogenesis of IBMPFD [10,[116][117][118] (for a recent review see [119]).…”

“…Zhang et al identified a region of about 25 unstructured amino acids in p47 that is not involved in p97 binding and is missing in p37, which is responsible for the inhibitory effect of p47. In the D1 domain of each p97 monomer two ADP-bound states exist in a dynamic equilibrium: the ADP-locked state containing prebound, difficult-to-remove ADP [28,114], and the ADP-open state where ADP has a reduced affinity to the D1 site and is ready to be exchanged [29]. In the wild-type protein, the equilibrium is in favor of the ADP-locked state, whereas in IBMPFD mutants (see below), the ADP-open state is prevalent.…”

“…Finally, SAXS structures were derived for the same nucleotide states [28]. In contrast to full-length p97, the N-D1 fragment appears to be more amenable to high resolution structural analysis and has therefore been extensively investigated [25,29,30], including structures of N-D1 variants carrying IBMPFD-associated point mutations [29,30] (see below).…”