**7. Implications concerning p97 function and disease**

One major contribution of p97 to cellular processes is its apparent participation in protein quality control and homeostasis, involving the ubiquitin-proteasome degradation pathway, ER associated degradation, and formation of autophagosomes. The multifaceted clinical presentation of patients with IBMPFD is consistent with the broad spectrum of p97 functions. Indeed, pathology and the cellular hallmarks such as the accumulation of inclusion bodies and rimmed vacuoles of IBMPFD can be reproduced both in cell culture and in animal models (Custer et al., 2010; Ju and Weihl, 2010; Weihl et al., 2006; Weihl et al., 2007). However, the structural and molecular basis of how p97 is involved in these different pathways and the mechanism of how p97 mutations lead to dysfunction remain elusive.

IBMPFD and p97, the Structural and Molecular Basis for Functional Disruption 167

defined by the binding of exchangeable ADP. 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 may represent this conformation. (4) Finally, there is the "Empty" state with nucleotide-binding sites unoccupied and the N-domain in an unknown position. For wild type p97, the transition between the "ADP-locked" and "ADPopen" states is thought to be tightly controlled, resulting in rare ADP-open states, leading to asymmetry in the nucleotide binding and N-domain conformation in a hexameric p97.

Fig. 6. **Models for the N-domain movement in p97 during ATP cycle** Schematic diagram for the control of the N-domain conformation in the wild type and IBMPFD associated N-D1 fragment of p97. Different domains are colored and labeled. The IBMPFD mutations are represented by yellow circles. Four states are defined for each nucleotide-binding site in D1: *Empty, ATP, ADP-locked* and *ADP-open* states, as labeled. The type of nucleotide bound at the D1-domain is labeled. Each subunit of the hexameric p97 is assumed to operate

From structural and molecular characterizations, we can infer that the non-uniform movement of p97 is essential to its function. In order to generate the up-and-down movement of the N-domain in a non-uniform fashion, the "ADP-locked" sites needed to be

independently in this model.

#### **7.1 IBMPFD mutations produce subtle structural and functional alteration in p97**

Using structural, biophysical and biochemical approaches and through detailed comparative study of wild type and IBMPFD mutant p97, details of the molecular mechanisms of p97 at the most fundamental level are beginning to emerge. As a late onset disease, the IBMPFD mutations in p97 are not expected to dramatically disrupt cellular functions. Indeed, as shown from the cell biological, structural and biochemical data, all IBMPFD mutants (1) appear to have a normal phenotype at least in the early stage of life in cultured cells, in yeast, and in fruit flies, (2) do not have observable structural alterations in their constituent domains, as compared to the wild type, (3) can form proper hexameric ring structures, (4) have nucleotide-binding pockets indistinguishable from those of the wild type, and (5) are able to undergo nucleotide-driven conformational change in solution.

In spite of these similarities, subtle yet significant differences have also been detected in IBMPFD mutants, including (1) overall up-regulated ATPase activities, (2) ability to undergo uniform nucleotide-dependent N-domain conformational change that leads to its crystallization in the presence of ATPS, (3) lowered binding affinity toward ADP in the D1 domain, (4) less non-exchangeable pre-bound ADP in the D1-domain, and (5) subtle differences in binding of adaptor proteins.

#### **7.2 Asymmetry in p97 function**

Enigmatic observations concerning the failure of wild type p97 to crystallize in the presence of ATPS, yet being able to undergo nucleotide-dependent N-domain conformational change in solution suggest the functional importance of the non-uniform binding of nucleotides by p97 to the D1-domains and of the asymmetry in N-domain conformations among its six subunits. This asymmetry is a built-in property of wild type p97, as demonstrated by ITC and heat or urea denaturation experiments with characteristically prebound ADP at the D1-domain. Although a p97 hexamer is formed by six identical monomers, a fraction of the D1 sites is always pre-occupied by ADP, which is very difficult to release or exchange with other nucleotides. As a result, ATP in solution is only able to access the empty D1 sites, which drive the N-domain to the Up-conformation, whereas the N-domains remain in the Down-conformation for those subunits with pre-bound ADP. Although it is not yet clear how p97 maintains this asymmetry during its catalytic cycle, some level of communication must exist among subunits.

A model for the ATP cycle in the D1-domain and the corresponding N-domain conformation has been proposed by integrating the structural and biochemical data of wild type and mutant p97 (Fig. 6). The model proposes four nucleotide-binding states for the D1 domain. (1) There is the "ATP" state with ATP bound and the N-domain in the Upconformation. Crystallographic and X-ray scattering experiments support the existence of this state in subunits of both mutant and wild type p97. It should be noted that due to nonexchangeable, pre-bound ADP in a wild type p97 hexamer, not all subunits can bring their N-domains to the Up-conformation, even with an excess of ATP in solution. (2) There is an "ADP-locked" state with non-exchangeable, pre-bound ADP at the D1 site and the Ndomain in the Down-conformation. This state appears to be important for wild type p97 function and the pre-bound ADP is particularly difficult to release. The structure of the N-D1 fragment of wild type p97 may represent this conformation. (3) The "ADP-open" state is

Using structural, biophysical and biochemical approaches and through detailed comparative study of wild type and IBMPFD mutant p97, details of the molecular mechanisms of p97 at the most fundamental level are beginning to emerge. As a late onset disease, the IBMPFD mutations in p97 are not expected to dramatically disrupt cellular functions. Indeed, as shown from the cell biological, structural and biochemical data, all IBMPFD mutants (1) appear to have a normal phenotype at least in the early stage of life in cultured cells, in yeast, and in fruit flies, (2) do not have observable structural alterations in their constituent domains, as compared to the wild type, (3) can form proper hexameric ring structures, (4) have nucleotide-binding pockets indistinguishable from those of the wild type, and (5) are able to undergo nucleotide-driven conformational change in solution.

In spite of these similarities, subtle yet significant differences have also been detected in IBMPFD mutants, including (1) overall up-regulated ATPase activities, (2) ability to undergo uniform nucleotide-dependent N-domain conformational change that leads to its crystallization in the presence of ATPS, (3) lowered binding affinity toward ADP in the D1 domain, (4) less non-exchangeable pre-bound ADP in the D1-domain, and (5) subtle

Enigmatic observations concerning the failure of wild type p97 to crystallize in the presence of ATPS, yet being able to undergo nucleotide-dependent N-domain conformational change in solution suggest the functional importance of the non-uniform binding of nucleotides by p97 to the D1-domains and of the asymmetry in N-domain conformations among its six subunits. This asymmetry is a built-in property of wild type p97, as demonstrated by ITC and heat or urea denaturation experiments with characteristically prebound ADP at the D1-domain. Although a p97 hexamer is formed by six identical monomers, a fraction of the D1 sites is always pre-occupied by ADP, which is very difficult to release or exchange with other nucleotides. As a result, ATP in solution is only able to access the empty D1 sites, which drive the N-domain to the Up-conformation, whereas the N-domains remain in the Down-conformation for those subunits with pre-bound ADP. Although it is not yet clear how p97 maintains this asymmetry during its catalytic cycle,

A model for the ATP cycle in the D1-domain and the corresponding N-domain conformation has been proposed by integrating the structural and biochemical data of wild type and mutant p97 (Fig. 6). The model proposes four nucleotide-binding states for the D1 domain. (1) There is the "ATP" state with ATP bound and the N-domain in the Upconformation. Crystallographic and X-ray scattering experiments support the existence of this state in subunits of both mutant and wild type p97. It should be noted that due to nonexchangeable, pre-bound ADP in a wild type p97 hexamer, not all subunits can bring their N-domains to the Up-conformation, even with an excess of ATP in solution. (2) There is an "ADP-locked" state with non-exchangeable, pre-bound ADP at the D1 site and the Ndomain in the Down-conformation. This state appears to be important for wild type p97 function and the pre-bound ADP is particularly difficult to release. The structure of the N-D1 fragment of wild type p97 may represent this conformation. (3) The "ADP-open" state is

differences in binding of adaptor proteins.

some level of communication must exist among subunits.

**7.2 Asymmetry in p97 function** 

**7.1 IBMPFD mutations produce subtle structural and functional alteration in p97** 

defined by the binding of exchangeable ADP. 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 may represent this conformation. (4) Finally, there is the "Empty" state with nucleotide-binding sites unoccupied and the N-domain in an unknown position. For wild type p97, the transition between the "ADP-locked" and "ADPopen" states is thought to be tightly controlled, resulting in rare ADP-open states, leading to asymmetry in the nucleotide binding and N-domain conformation in a hexameric p97.

Fig. 6. **Models for the N-domain movement in p97 during ATP cycle** Schematic diagram for the control of the N-domain conformation in the wild type and IBMPFD associated N-D1 fragment of p97. Different domains are colored and labeled. The IBMPFD mutations are represented by yellow circles. Four states are defined for each nucleotide-binding site in D1: *Empty, ATP, ADP-locked* and *ADP-open* states, as labeled. The type of nucleotide bound at the D1-domain is labeled. Each subunit of the hexameric p97 is assumed to operate independently in this model.

From structural and molecular characterizations, we can infer that the non-uniform movement of p97 is essential to its function. In order to generate the up-and-down movement of the N-domain in a non-uniform fashion, the "ADP-locked" sites needed to be

IBMPFD and p97, the Structural and Molecular Basis for Functional Disruption 169

Bersano, A.; Del Bo, R.; Lamperti, C.; Ghezzi, S.; Fagiolari, G.; Fortunato, F.; Ballabio, E.;

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activated to the "ADP-open" state. It is thought that in wild type p97 control of the transition between the ADP-locked and ADP-open state in D1 could be achieved in two ways: (1) the binding of adaptor proteins to the N-domain, or (2) the hydrolysis of ATP in the D2-domain. The N-domain was shown to have an influence on the ATPase activity of both N-D1 and the full-length p97 ortholog, VAT, as the N-domain-deleted p97 mutants have higher ATPase activity (Gerega et al., 2005). The binding of adaptor protein p47 to the N-domain was shown to inhibit the ATPase activity of p97 (Meyer et al., 1998). Communication between D1 and D2 is also known to exist for p97 and other type II AAA+ proteins. For example, it was shown that the absence of D2-domain inhibits the nucleotide exchange activity in D1 (Davies et al., 2005). The yeast Hsp104, another type II AAA+ protein, displays cooperative kinetics and inter-domain communication for its two ATPase domains (Hattendorf and Lindquist, 2002). However, the exact details of these possible control mechanisms for the switching of D1 nucleotide states remain elusive. Like many AAA+ proteins involved in protein quality control such as *E. coli* ClpA and yeast Hsp104, p97 functions in handling protein substrates to various pathways, which requires the presence of the N-domain. Although how p97 handles these substrates has yet to be defined, one advantage of asymmetric interaction over symmetric seems that the former ensures continuous contacts with the substrates.

#### **7.3 Loss of asymmetry in IBMPFD mutant p97**

Mapping the IBMPFD mutations to the Down-conformation of p97 reveals the clustering of these mutations at the interface between the N- and D1-domains. Site-directed mutagenesis of R86A, a residue present at the N-D1 interface but not identified as an IBMPFD mutation, showed to possess all the structural and biochemical characteristics of an IBMPFD mutant p97 (Tang et al., 2010). This suggests that the N-D1 interface residues are critical for the proper function of p97 by providing tight regulation of the movement of the N-domain and the nucleotide state of the D1-domain.

Instead of prominent structural changes, IBMPFD mutations introduce subtle modifications to p97, apparently disrupting communication among the monomers. Unlike the wild type p97, IBMPFD mutants allow easy displacement of pre-bound ADP at the D1-domain by ATPS, resulting in a unified nucleotide state, and hence, a symmetric hexamer in the Upconformation. Using the same ATP catalytic cycle model for the D1-domain shown above, it was postulated that the difference between the wild type and mutants lies in the transition between the "ADP-locked" state and the "ADP-open" state. While this transition is tightly regulated in wild type p97, this control mechanism is altered in IBMPFD mutants, leading to a high concentration of subunits in the "ADP-open" state (Fig. 6). Consequently, p97 mutants undergo a uniform N-domain conformational change in response to high concentrations of ATPS, leading to a defective enzyme.

#### **8. References**

Allen, M.D.; Buchberger, A. & Bycroft, M. (2006). The PUB domain functions as a p97 binding module in human peptide N-glycanase. *J Biol Chem,* Vol.281, No.35, (Sep 1), pp. 25502-25508

activated to the "ADP-open" state. It is thought that in wild type p97 control of the transition between the ADP-locked and ADP-open state in D1 could be achieved in two ways: (1) the binding of adaptor proteins to the N-domain, or (2) the hydrolysis of ATP in the D2-domain. The N-domain was shown to have an influence on the ATPase activity of both N-D1 and the full-length p97 ortholog, VAT, as the N-domain-deleted p97 mutants have higher ATPase activity (Gerega et al., 2005). The binding of adaptor protein p47 to the N-domain was shown to inhibit the ATPase activity of p97 (Meyer et al., 1998). Communication between D1 and D2 is also known to exist for p97 and other type II AAA+ proteins. For example, it was shown that the absence of D2-domain inhibits the nucleotide exchange activity in D1 (Davies et al., 2005). The yeast Hsp104, another type II AAA+ protein, displays cooperative kinetics and inter-domain communication for its two ATPase domains (Hattendorf and Lindquist, 2002). However, the exact details of these possible control mechanisms for the switching of D1 nucleotide states remain elusive. Like many AAA+ proteins involved in protein quality control such as *E. coli* ClpA and yeast Hsp104, p97 functions in handling protein substrates to various pathways, which requires the presence of the N-domain. Although how p97 handles these substrates has yet to be defined, one advantage of asymmetric interaction over symmetric seems that the former ensures

Mapping the IBMPFD mutations to the Down-conformation of p97 reveals the clustering of these mutations at the interface between the N- and D1-domains. Site-directed mutagenesis of R86A, a residue present at the N-D1 interface but not identified as an IBMPFD mutation, showed to possess all the structural and biochemical characteristics of an IBMPFD mutant p97 (Tang et al., 2010). This suggests that the N-D1 interface residues are critical for the proper function of p97 by providing tight regulation of the movement of the N-domain and

Instead of prominent structural changes, IBMPFD mutations introduce subtle modifications to p97, apparently disrupting communication among the monomers. Unlike the wild type p97, IBMPFD mutants allow easy displacement of pre-bound ADP at the D1-domain by ATPS, resulting in a unified nucleotide state, and hence, a symmetric hexamer in the Upconformation. Using the same ATP catalytic cycle model for the D1-domain shown above, it was postulated that the difference between the wild type and mutants lies in the transition between the "ADP-locked" state and the "ADP-open" state. While this transition is tightly regulated in wild type p97, this control mechanism is altered in IBMPFD mutants, leading to a high concentration of subunits in the "ADP-open" state (Fig. 6). Consequently, p97 mutants undergo a uniform N-domain conformational change in response to high

Allen, M.D.; Buchberger, A. & Bycroft, M. (2006). The PUB domain functions as a p97

binding module in human peptide N-glycanase. *J Biol Chem,* Vol.281, No.35, (Sep

continuous contacts with the substrates.

the nucleotide state of the D1-domain.

1), pp. 25502-25508

**8. References** 

**7.3 Loss of asymmetry in IBMPFD mutant p97** 

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**10** 

*1Japan 2USA* 

**Congenital Myasthenic Syndromes –** 

Kinji Ohno1, Mikako Ito1 and Andrew G. Engel2

*Nagoya University Graduate School of Medicine, Nagoya, 2Department of Neurology, Mayo Clinic, Rochester, Minnesota,* 

**Molecular Bases of Congenital Defects** 

*1Division of Neurogenetics, Center for Neurological Diseases and Cancer,* 

**of Proteins at the Neuromuscular Junction** 

Congenital myasthenic syndromes (CMS) are heterogeneous disorders caused by mutations in molecules expressed at the neuromuscular junction (NMJ) (Fig. 1). Each mutation affects the expression level or the functional properties or both of the mutant molecule. No fewer than 11 defective molecules at the NMJ have been identified to date. The mutant molecules include (i) acetylcholine receptor (AChR) subunits that forms nicotinic AChR and generate endplate potentials (Ohno *et al.*, 1995; Sine *et al.*, 1995), (ii) rapsyn that anchors and clusters AChRs at the endplate (Ohno *et al.*, 2002; Milone *et al.*, 2009), (iii) agrin that is released from nerve terminal and induces AChR clustering by stimulating the downstream LRP4/MuSK/Dok-7/rapsyn/AChR pathway (Huze *et al.*, 2009), (iv) muscle-specific receptor tyrosine kinase (MuSK) that transmits the AChR-clustering signal from agrin/LRP4 to Dok-7/rapsyn/AChR (Chevessier *et al.*, 2004; Chevessier *et al.*, 2008), (v) Dok-7 that interacts with MuSK and exerts the AChR-clustering activity (Beeson *et al.*, 2006; Hamuro *et al.*, 2008), (vi) plectin that is an intermediate filament-associate protein concentrated at sites of mechanical stress (Banwell *et al.*, 1999; Selcen *et al.*, 2011), (vii) glutamine-fructose-6 phosphate aminotransferase 1 encoded by *GFPT1*, the function of which at the NMJ has not been elucidated (Senderek *et al.*, 2011), (viii) skeletal muscle sodium channel type 1.4 (NaV1.4) that spreads depolarization potential from endplate throughout muscle fibers (Tsujino *et al.*, 2003), (ix) collagen Q that anchors acetylcholinesterase (AChE) to the synaptic basal lamina (Ohno *et al.*, 1998; Ohno *et al.*, 1999; Kimbell *et al.*, 2004), (x) 2-laminin that forms a cruciform heterotrimeric lamins-221, -421, and -521 and links extracellular matrix molecules to the -dystroglycan at the NMJ (Maselli *et al.*, 2009), (xi) choline acetyltransferase (ChAT) that resynthesizes acetylcholine from recycled choline at the nerve terminal (Ohno *et al.*, 2001). AChR (Lang & Vincent, 2009), MuSK (Hoch *et al.*, 2001; Cole *et al.*, 2008), and LRP4 (Higuchi *et al.*, 2011) are also targets of myasthenia gravis, in which

autoantibody against each molecule impairs the neuromuscular transmission.

CMS are classified into three groups of postsynaptic, synaptic, and presynaptic depending on the localization of the defective molecules. Among the eleven molecules introduced

**1. Introduction** 


*2USA* 
