AAA Proteins, Their Functions and Related Diseases

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Our recent work has been focusing on two mammalian AAA proteins: the human AAA protein p97 and the mouse mitochondrial AAA protein bcs1. The human p97 is a major cytosolic AAA chaperone. Although it has been known that D2 ring of p97 contributes most to the overall ATPase activity of p97, the function of the D1 ring is not clear. Our work has contributed significantly to our understanding the function of the D1 ring, which is the regulatory domain of p97. We focus our study on one type of p97 mutants that cause IBMPFD or MSP1. IBMPFD mutants have single amino acid substitutions at the interface between the N-terminal domain (N-domain) and the adjacent AAA domain (D1) and our work suggests that the mutations result in a reduced affinity for ADP. The structures of p97 N-D1 fragments bearing IBMPFD mutations adopt an Up N-domain conformation or Up-conformation in the presence of Mg2+-ATPgS, which is reversible by ADP (Down-conformation), demonstrating for the first time the nucleotide-dependent conformational change of the N-domain. We further found that wild type p97 also undergoes nucleotide-dependent Up- and Down-N-domain conformational change in solution. 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 reports of the number of occluded ADP in wild-type p97. By contrast, mutant p97 N-D1 fragments displayed reduced binding affinities for ADP. For example, the R155H mutant showed a maximum reduction with a Kd of 4.25 uM. Notably, the number of occluded ADP in mutant p97 is dramatically reduced. Unexpectedly, the titration profiles with ATPgS 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 ATPgS titration experiments. A model with four nucleotide-binding states for the ATP cycle in the D1-domain was proposed. We also investigated how IBMPFD mutations affect the molecular mechanism that governs the function of p97. We showed that within the hexameric ring of a mutant p97, D1 domains fail to regulate their respective nucleotide-binding states, as evidenced by the lower amount of prebound ADP, weaker ADP binding affinity, full occupancy of ATP-gS binding, and elevated overall ATPase activity, indicating a loss of communication among subunits. Defective communication between subunits is further illustrated by altered conformation in the side chain of residue Phe-360 that probes into the nucleotide-binding pocket from a neighboring subunit. Consequently, conformations of N-domains in a hexameric ring of a mutant p97 become uncoordinated, thus impacting its ability to process substrate. Our investigation into the intra-molecular communication pathway also led to the discovery that the presence of a 22 amino acid peptide at the end of N-D1 truncate, named D1-D2 linker, of the human AAA+ protein p97 has been shown to activate ATP hydrolysis of the D1 domain, but the mechanism of activation remains unclear. We identified the N-terminal half of this D1-D2 linker, which is ubiquitously conserved from human to fungi, is essential for the activation of the ATPase. Based on the analysis of all available p97 structures, we observed that the presence of the D1-D2 linker affects the way subunits of p97 associate to form hexameric rings, which was manifested in the crystal symmetry. The presence of the linker leads to lower crystal symmetry, an observation that is reinforced by the two new crystal structures, a wild-type N-D1 truncate with the linker and a L198W mutant N-D1 truncate without the linker, determined in the present work. The lack of activity of the D1 ATPase domain in the absence of D1-D2 linker implies the functional importance of asymmetric subunit arrangement, which we suggest to be estimated quantitatively by the metrics Asymmetirc Index. Structure comparison correlates the conformation of the D1-D2 linker to conformation of the Arg-finger from a neighboring subunit, suggesting a regulatory role of the D1-domain in the conformation of D2-domain. More recently, we studied the association of cytosolic AAA protein p97 to membranes, which is essential for various cellular processes including the endoplasmic reticulum (ER)-associated degradation. The N-domain of p97 is known for undergoing large nucleotide-dependent conformational change but the physiological relevance this conformational change has not been established. We showed p97 is recruited to the ER membrane predominantly by interacting with VIMP, an ER resident protein. The recruitment can be regulated through a nucleotide-dependent conformation switch of the N-domain in wild-type p97 and this regulation is obliterated in pathogenic mutants. The molecular mechanism of the regulation is revealed by a series of structures of p97, VIMP and their complex, thus suggesting a physiological role of the nucleotide-dependent conformational change of the N-domain of p97. In addition, intermediate positions of the N-domain are seen when AMP-PNP occupies the D1-domain, allowing construction of a trajectory for the N-domain movement. Our findings suggest the nucleotide-dependent membrane interaction cycle may be applicable to other p97-dependent events. Another AAA protein that are being actively pursued in the lab is called bcs1 that, unlike the functions of most AAA proteins known to date, involves in folded protein translocation across the membrane. Having determined the structures of mouse Bcs1 (mBcs1) in different nucleotide states and conformations, we now have acquired a structural framework from which more detailed mechanistic insights into the transport mechanism of Bcs1 can be expected. Currently, we focus on studies that will likely reveal how Bcs1 recognizes and binds the folded ISP-ED, capture its action in translocating the substrate across the membrane, and visualize how it releases the substrate into the membrane. To achieve these goals, a combination of various research approaches will have to be employed. From a structural point of view, it is necessary to obtain the structure of Bcs1 in complex with the substrate ISP in order to address the questions on how substrate binding trigger changes in Bcs1 and whether binding of substrate is sufficient to induce nucleotide exchange. Structures are also needed to determine whether subunits of Bcs1 functions in a sequential fashion or in a concerted manner. The former is the hallmark of the hand-over-hand or split wash mechanism of translocation displayed by many hexameric AAA proteins. In the apo and ADP bound structures, the unknown density plugging the small pore in the center of the Bcs1-specific domains should also be investigated. Biochemically, kinetic study of the life span of different nucleotide states will provide clues on the rate limiting steps in the reaction landscape. Coupling these studies with mutagenesis will likely play a major role in verifying various mechanistic hypotheses. For example, to prevent proton leakage during translocation, an airlock-like mechanism was proposed. However, how the opening and closure of the seal pore is controlled requires further elucidation. Mutagenesis studies will allow functional and structural characterizations of many documented disease-related mutants. The structures should also facilitate development of drugs to modulate function of Bcs1.