Until now, there are 13 positions in p97 where IBMPD mutations have been identified, all of which were found located at the N-D1 interface. Several IBMPFD mutants were expressed and purified (Table 2.1). Since all IBMPFD mutations are located in the N- or D1-domain of p97 in the primary sequence and to facilitate crystallization, we expressed and purified a number of N-D1 fragments of p97 mutants, crystallized some of them and obtained five structures under various nucleotide bound conditions. In these structures, all mutations are located on the surface of various domains and there are no significant changes in each domain due to the mutations when compared to their wild type counterparts. In all mutant structures, six N-D1 protomers form a ring-shaped hexamer through interactions between D1-domains with each N-domain attached to its cognate D1 domain. In contrast to all previously reported p97 structures with ADP bound in the D1-domains, we found that ATPgamaS can be incorporated into the D1-domain of all crystal structures of IBMPFD mutants. Also new to the p97 structure is the presence of a Mg2+ ion in the nucleotide-binding site of every subunit, which was not observed in previously reported structures. The Mg2+ ion is at the center of an octahedral mer-triaquo complex with the additional three oxo ligands coming from the highly conserved Thr252 and from the beta- and gama-phosphates. When the structures of the hexameric D1 rings of mutants with Mg2+.ATPgamaS bound were aligned with the ADP-bound wild type p97 N-D1, the D1 rings superimpose nicely, whereas the N-domains display dramatic conformational changes by swinging upward by 13 A (Up-conformation);this movement of the N-domain includes a large rotation of 93 degree. The upward movement of N-domains is a consequence of ATPgamaS binding and cannot be induced by ADP binding. This result establishes the interdependency between the conformation of the N-domain and the nucleotide state in the D1-domain for IBMPFD mutants. Moreover, mutant p97 hexamers display a concerted N-domain conformational movement. Part of the N-D1 linker undergoes a coil-to-helix transition, as the nucleotide of p97 switches from ADP to ATPgamaS. Wild type and mutant N-D1 fragments were also studied in the presence of ATPgamaS or ADP by SAXS. The radii of gyration (Rg) are consistently 3-5 A smaller for the ATPgamaS-bound N-D1 fragment as compared to the ADP-bound form. The conformational change of N-D1 in solution can also be demonstrated by the distance distribution functions, p(r), in which a significant shift in the distribution towards shorter vectors was observed for the ATPgamaS-bound N-D1 fragments, This shift in p(r) is most obvious at vector lengths beyond 90 A, consistent with the large-scale N-domain conformational change. Furthermore, calculated changes in the distribution function based on crystal structures are in agreement with the experimentally obtained distribution functions, suggesting that the crystallographically observed differences in conformation of the N-domain exist in solution not only for p97 mutants but also for wild type p97. Using isothermal titration calorimetry (ITC), we determined a Kd value of 0.88 uM towards ADP for the wild type N-D1 with a stoichiometry of 0.35, suggesting only 2 out of 6 sites are available for binding, which is consistent with previously reported values. By contrast, mutant p97 N-D1 fragments displayed reduced binding affinities for ADP and the level of reduction is site dependent. For example, the R155H mutant showed a maximum reduction with a Kd of 4.25 uM. Notably, the changes in the binding stoichiometry are correlated with the changes in binding affinities for the mutants. Consistent with the previous findings, wild type p97 showed a Kd value for ATPgamaS of 0.89 uM, similar to that for ADP. Unexpectedly, the titration profiles with ATPgamaS for mutants were biphasic and can only be fitted to a two-site model. The Kd values for the high affinity site were well determined and close to 0.1 uM for all mutants, whereas those for the low affinity site were associated with significant errors. Again, mutant p97 displayed higher stoichiometry than wild type in the ATPgamaS titration experiments. A model with four nucleotide-binding states for the ATP cycle in the D1-domain was proposed. First, there is an """"""""ATP"""""""" state, with ATP bound and the N-domain in the Up-conformation. In a wild type p97 hexamer, due to non-exchangeable, pre-bound ADP, not all subunits will have their N-domains in the Up-conformation even with an excess amount of ATP in solution. We therefore hypothesize that there is an """"""""ADP-locked"""""""" state, with non-exchangeable, pre-bound ADP at the D1 site and the N-domain in the Down-conformation. This state appears to be important for wild type p97 function and the pre-bound ADP is particularly difficult to exchange. The structure of the N-D1 fragment of wild type p97 may represent this conformation. In a third state, termed """"""""ADP-open"""""""", ADP is bound but exchangeable. This state was observed for mutant p97 by its biphasic ITC titration profile and is presumably in equilibration with the """"""""ADP-locked"""""""" state. The structure of R155H with bound ADP represents this conformation. The fourth state is the """"""""Empty"""""""" state, with nucleotide-binding sites unoccupied and the N-domain in an unknown position. The difference between the wild type and mutants, however, lies in the transition between the ADP-locked state and the ADP-open state. We propose that in the wild type protein this transition is tightly controlled and characterized by the asymmetry in nucleotide binding states in D1-domains of different subunits, resulting in a low concentration of the ADP-open state, whereas in IBMPFD mutants, this control mechanism is altered, leading to a high concentration of subunits in the ADP-open state.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010600-08
Application #
8349062
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2011
Total Cost
$225,803
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Xia, Di; Tang, Wai Kwan; Ye, Yihong (2016) Structure and function of the AAA+ ATPase p97/Cdc48p. Gene 583:64-77
Tang, Wai Kwan; Xia, Di (2016) Role of the D1-D2 Linker of Human VCP/p97 in the Asymmetry and ATPase Activity of the D1-domain. Sci Rep 6:20037
Xia, Di; Ye, Yihong (2015) In Search of a Cure for Proteostasis-Addicted Cancer: A AAA Target Revealed. Cancer Cell 28:550-2
Tang, Wai Kwan; Xia, Di (2013) Altered intersubunit communication is the molecular basis for functional defects of pathogenic p97 mutants. J Biol Chem 288:36624-35
Ma, Jichun; Tang, Wai Kwan; Esser, Lothar et al. (2012) Characterization of crystals of an antibody-recognition fragment of the cancer differentiation antigen mesothelin in complex with the therapeutic antibody MORAb-009. Acta Crystallogr Sect F Struct Biol Cryst Commun 68:950-3
Ma, Jichun; Tang, Wai Kwan; Esser, Lothar et al. (2012) Recognition of mesothelin by the therapeutic antibody MORAb-009: structural and mechanistic insights. J Biol Chem 287:33123-31
Tang, Wai Kwan; Xia, Di (2012) Structural and functional deviations in disease-associated p97 mutants. J Struct Biol 179:83-92
Tang, Wai Kwan; Li, Dongyang; Li, Chou-chi et al. (2010) A novel ATP-dependent conformation in p97 N-D1 fragment revealed by crystal structures of disease-related mutants. EMBO J 29:2217-29