Derlin-1 Regulates Mutant VCP-Linked Pathogenesis and Endoplasmic Reticulum Stress-Induced Apoptosis

Mutations in VCP (Valosin-containing protein), an AAA ATPase critical for ER-associated degradation, are linked to IBMPFD (Inclusion body myopathy with Paget disease and frontotemporal dementia). Using a Drosophila IBMPFD model, we have identified the ER protein Derlin-1 as a modifier of pathogenic TER94 (the fly VCP homolog) mutants. Derlin-1 binds to TER94 directly, and this interaction is essential for Derlin-1 overexpression to suppress the pathogenic TER94-induced neurodegeneration. Derlin-1 overexpression reduces the elevated ATPase activity of pathogenic TER94, implying that IBMPFD is caused by ATPase hyper-activation. Under physiological condition, Derlin-1 expression is increased upon ER stress to recruit TER94 to the ER. However, in response to severe ER stress, Derlin-1 is required for activating apoptosis to eliminate damaged cells. This pro-apoptotic response is mimicked by Derlin-1 overexpression, which elicits acute ER stress and triggers apoptosis via a novel C-terminal motif (α). As this Derlin-1-dependent cell death is negated by TER94 overexpression, we propose that while Derlin-1 and VCP work cooperatively in ER stress response, their imbalance has a role in removing cells suffering prolonged ER stress.     

Proteomic analysis of a drosophila IBMPFD model reveals potential pathogenic mechanisms

IBMPFD, Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia, is a hereditary degenerative disorder due to single missense mutations in VCP (Valosin-Containing Protein). The mechanisms of how mutations of VCP lead to IBMPFD remain mysterious. Here we utilize two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry to study the IBMPFD disorder at the protein level. With this set-up, we are able to employ comparative proteomics to analyze IBMPFD disease using Drosophila melanogaster as our disease model organism. Head proteome of transgenic D. melanogaster expressing wild type VCP is compared, respectively, with the head proteome of transgenic mutant type VCPs that correspond to human IBMPFD disease alleles (TER94(A229E), TER94(R188Q), and TER94(R152H)). Of all the proteins identified, a significant fraction of proteins altered in TER94(A229E) and TER94(R188Q) mutants belong to the same functional categories, i.e. apoptosis and metabolism. Among these, Drosophila transferrin is observed to be significantly up-regulated in mutant flies expressing TER94(A229E). A knock-down experiment suggests that fly transferrin might be a potential modifier in IBMPFD disease. The molecular analysis of IBMPFD disease may benefit from the proteomics approach which combines the advantages of high throughput analysis and the focus on protein levels.     

Impaired protein aggregate handling and clearance underlie the pathogenesis of p97/VCP-associated disease

Mutations in p97/VCP cause the multisystem disease inclusion body myopathy, Paget disease of the bone and frontotemporal dementia (IBMPFD). p97/VCP is a member of the AAA+ (ATPase associated with a variety of activities) protein family and has been implicated in multiple cellular processes. One pathologic feature in IBMPFD is ubiquitinated inclusions, suggesting that mutations in p97/VCP may affect protein degradation. The present study shows that IBMPFD mutant expression increases ubiquitinated proteins and susceptibility to proteasome inhibition. Co-expression of an aggregate prone protein such as expanded polyglutamine in IBMPFD mutant cells results in an increase in aggregated protein that localizes to small inclusions instead of a single perinuclear aggresome. These small inclusions fail to co-localize with autophagic machinery. IBMPFD mutants avidly bind to these small inclusions and may not allow them to traffic to an aggresome. This is rescued by HDAC6, a p97/VCP-binding protein that facilitates the autophagic degradation of protein aggregates. Expression of HDAC6 improves aggresome formation and protects IBMPFD mutant cells from polyglutamine-induced cell death. Our study emphasizes the importance of protein aggregate trafficking to inclusion bodies in degenerative diseases and the therapeutic benefit of inclusion body formation.     

Rescue of growth defects of yeast cdc48 mutants by pathogenic IBMPFD-VCPs

VCP/p97/Cdc48 is a hexameric ring-shaped AAA ATPase that participates in a wide variety of cellular functions. VCP is a very abundant protein in essentially all types of cells and is highly conserved among eukaryotes. To date, 19 different single amino acid-substitutions in VCP have been reported to cause IBMPFD (inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia), an autosomal dominant inherited human disease. Moreover, several similar single amino acid substitutions have been proposed to associate with a rare subclass of familial ALS. The mechanisms by which these mutations contribute to the pathogenesis are unclear. To elucidate potential functional differences between wild-type and pathogenic VCPs, we expressed both VCPs in yeast cdc48 mutants. We observed that all tested pathogenic VCPs suppressed the temperature-sensitive phenotype of cdc48 mutants more efficiently than wild-type VCP. In addition, pathogenic VCPs, but not wild-type VCP, were able to rescue a lethal cdc48 disruption. In yeast, pathogenic VCPs, but not wild-type VCP, formed apparent cytoplasmic foci, and these foci were transported to budding sites by the Myo2/actin-mediated transport machinery. The foci formation of pathogenic VCPs appeared to be associated with their suppression of the temperature-sensitive phenotype of cdc48 mutants. These results support the idea that the pathogenic VCP mutations create dominant gain-of-functions rather than a simple loss of functional VCP. Their unique properties in yeast could provide a convenient drug-screening system for the treatment of these diseases.     

Identification of ter94, Drosophila VCP, as a modulator of polyglutamine-induced neurodegeneration

We have successfully generated a Drosophila model of human polyglutamine (polyQ) diseases by the targeted expression of expanded-polyQ (ex-polyQ) in the Drosophila compound eye. The resulting eye degeneration is progressive and ex-polyQ dosage- and ex-polyQ length-dependent. Furthermore, intergenerational changes in repeat length were observed in homozygotes, with concomitant changes in the levels of degeneration. Through genetic screening, using this fly model, we identified loss-of-function mutants of the ter94 gene that encodes the Drosophila homolog of VCP/CDC48, a member of the AAA+ class of the ATPase protein family, as dominant suppressors. The suppressive effects of the ter94 mutants on ex-polyQ-induced neurodegeneration correlated well with the degrees of loss-of-function, but appeared not to result from the inhibition of ex-polyQ aggregate formation. In the ex-polyQ-expressing cells of the late pupa, an upregulation of ter94 expression was observed prior to cell death. Co-expression of ter94 with ex-polyQ severely enhanced eye degeneration. Interestingly, when ter94 was overexpressed in the eye by increasing the transgene copies, severe eye degeneration was induced. Furthermore, genetical studies revealed that ter94 was not involved in grim-, reaper-, hid-, ced4-, or p53-induced cell death pathways. From these observations, we propose that VCP is a novel cell death effector molecule in ex-polyQ-induced neurodegeneration, where the amount of VCP is critical. Control of VCP expression may thus be a potential therapeutic target in ex-polyQ-induced neurodegeneration.     

VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration

Neuronal cell death, abnormal protein aggregates, and cytoplasmic vacuolization are major pathologies observed in many neurodegenerative disorders such as the polyglutamine (polyQ) diseases, prion disease, Alzheimer disease, and the Lewy body diseases, suggesting common mechanisms underlying neurodegeneration. Here, we have identified VCP/p97, a member of the AAA+ family of ATPase proteins, as a polyQ-interacting protein in vitro and in vivo, and report on its characterization. Endogenous VCP co-localized with expanded polyQ (ex-polyQ) aggregates in cultured cells expressing ex-polyQ, with nuclear inclusions in Huntington disease patient brains, and with Lewy bodies in patient samples. Moreover, the expression of VCP mutants with mutations in the 2nd ATP binding domain created cytoplasmic vacuoles, followed by cell death. Very similar vacuoles were also induced by ex-polyQ expression or proteasome inhibitor treatment. These results suggest that VCP functions not only as a recognition factor for abnormally folded proteins but also as a pathological effector for several neurodegenerative phenotypes. VCP may thus be an ideal molecular target for the treatment of neurodegenerative disorders.     

Valosin-containing protein (VCP): structure,functions, and implications in neurodegenerative diseases

Valosin-containing protein (VCP) is a hexameric protein belonging to the type II AAA+ (ATPases Associated with diverse cellular Activities) protein family. VCP governs multiple cellular processes and its diverse functions are determined by its interaction with a wide variety of partners and cofactors. Recently, mutations in VCP were suggested to cause inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, amyotrophic lateral sclerosis, and Huntington’s disease. However, the pathogenic mechanisms of VCP mutations in these diseases are still largely unknown. In this review, we summarize the structure and cellular functions of VCP, especially focusing on apoptosis and two major cellular degradation pathways, the ubiquitin–proteasome system and autophagy. We also list the representative VCP mutations and discuss their potential association with neurodegenerative diseases.     

Functional role of post-translational modifications of Sp1 in tumorigenesis

Both Neurofibromatosis type I (NF1) and inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) are autosomal dominant genetic disorders. These two diseases are fully penetrant but with high heterogeneity in phenotypes, suggesting the involvement of genetic modifiers in modulating patients' phenotypes. Although NF1 is recognized as a developmental disorder and IBMPFD is associated with degeneration of multiple tissues, a recent study discovered the direct protein interaction between neurofibromin, the protein product of the NF1 gene, and VCP/p97, encoded by the causative gene of IBMPFD. Both NF1 and VCP/p97 are critical for dendritic spine formation, which provides the cellular mechanism explaining the cognitive deficits and dementia found in patients. Moreover, disruption of the interaction between neurofibromin and VCP impairs dendritic spinogenesis. Neurofibromin likely influences multiple downstream pathways to control dendritic spinogenesis. One is to activate the protein kinase A pathway to initiate dendritic spine formation; another is to regulate the synaptic distribution of VCP and control the activity of VCP in dendritic spinogenesis. Since neurofibromin and VCP/p97 also regulate cell growth and bone metabolism, the understanding of neurofibromin and VCP/p97 in neurons may be applied to study of cancer and bone. Statin treatment rescues the spine defects caused by VCP deficiency, suggesting the potential role of statin in clinical treatment for these two diseases.     

The Drosophila homologue of the hereditary spastic paraplegia protein, spastin, severs and disassembles microtubules

Hereditary spastic paraplegias (HSPs), a group of neurodegenerative disorders characterized by lower-extremity spasticity and weakness, are most commonly caused by mutations in the spastin gene, which encodes a AAA+ ATPase related to the microtubule-severing protein katanin. A Drosophila homolog of spastin (D-spastin) was identified recently, and D-spastin RNAi-treated or genetic null flies show neurological defects, and protein overexpression decreases the density of cellular microtubules. Elucidating spastin's function and disease mechanism will require a more detailed understanding of its structure and biochemical mechanism. Here, we have investigated the effects of D-spastin, individual D-spastin domains, and D-spastin proteins bearing disease mutations on microtubules in cellular and in vitro assays. We show that D-spastin, like katanin, displays ATPase activity and uses energy from ATP hydrolysis to sever and disassemble microtubules; disease mutations abolish or partially interfere with these activities.     

From neurodevelopment to neurodegeneration: the interaction of neurofibromin and valosin-containing protein/p97 in regulation of dendritic spine formation

Both Neurofibromatosis type I (NF1) and inclusion body myopathy with Paget''s disease of bone and frontotemporal dementia (IBMPFD) are autosomal dominant genetic disorders. These two diseases are fully penetrant but with high heterogeneity in phenotypes, suggesting the involvement of genetic modifiers in modulating patients'' phenotypes. Although NF1 is recognized as a developmental disorder and IBMPFD is associated with degeneration of multiple tissues, a recent study discovered the direct protein interaction between neurofibromin, the protein product of the NF1 gene, and VCP/p97, encoded by the causative gene of IBMPFD. Both NF1 and VCP/p97 are critical for dendritic spine formation, which provides the cellular mechanism explaining the cognitive deficits and dementia found in patients. Moreover, disruption of the interaction between neurofibromin and VCP impairs dendritic spinogenesis. Neurofibromin likely influences multiple downstream pathways to control dendritic spinogenesis. One is to activate the protein kinase A pathway to initiate dendritic spine formation; another is to regulate the synaptic distribution of VCP and control the activity of VCP in dendritic spinogenesis. Since neurofibromin and VCP/p97 also regulate cell growth and bone metabolism, the understanding of neurofibromin and VCP/p97 in neurons may be applied to study of cancer and bone. Statin treatment rescues the spine defects caused by VCP deficiency, suggesting the potential role of statin in clinical treatment for these two diseases.     

D1 ring is stable and nucleotide-independent,whereas D2 ring undergoes major conformational changes during the ATPase cycle of p97-VCP

The 97-kDa valosin-containing protein (p97-VCP) belongs to the AAA (ATPases associated with various cellular activities) family and acts as a molecular chaperone in diverse cellular events, including ubiquitinproteasome-mediated degradation. We previously showed that VCP contains a substrate-binding domain, N, and two conserved ATPase domains, D1 and D2, of which D2 is responsible for the major enzyme activity. VCP has a barrel-like structure containing two stacked homo-hexameric rings made of the D1 and D2 domains, and this structure is essential for its biological functions. During ATPase cycles, VCP undergoes conformational changes that presumably apply tensions to the bound substrate, leading to the disassembly of protein complexes or unfolding of the substrate. How ATPase activity is coupled with the conformational changes in VCP complex and the D1 and D2 rings is not clear. In this report, we took biochemical approaches to study the structure of VCP in different nucleotide conditions to depict the conformational changes in the ATPase cycles. In contrast to many AAA chaperones that require ATP/ADP to form oligomers, both wild type VCP and ATP-binding site mutants can form hexamers without the addition of nucleotide. This nucleotide-independent hexamerization requires an intact D1 and the down-stream linker sequence of VCP. Tryptophan fluorescence and trypsin digestion analyses showed that ATP/ADP binding induces dramatic conformational changes in VCP. These changes do not require the presence of an intact ATP-binding site in D1 and is thus mainly attributed to the D2 domain. We propose a model whereby D1, although undergoing minor conformational changes, remains as a relatively trypsin-resistant hexameric ring throughout the ATPase cycle, whereas D2 only does so when it binds to ATP or ADP. After ADP is released at the end of the ATP hydrolysis, D2 ring is destabilized and adopts a relatively flexible and open structure.     

Exome sequencing reveals VCP mutations as a cause of familial ALS

Using exome sequencing, we identified a p.R191Q amino acid change in the valosin-containing protein (VCP) gene in an Italian family with autosomal dominantly inherited amyotrophic lateral sclerosis (ALS). Mutations in VCP have previously been identified in?families with Inclusion Body Myopathy, Paget disease, and Frontotemporal Dementia (IBMPFD). Screening of VCP in a cohort of 210 familial ALS cases and 78 autopsy-proven ALS cases identified four additional mutations including a p.R155H mutation in a pathologically proven case of ALS. VCP protein is essential for maturation of ubiquitin-containing autophagosomes, and mutant VCP toxicity is partially mediated through its effect on TDP-43 protein, a major constituent of ubiquitin inclusions that neuropathologically characterize ALS. Our data broaden the phenotype of IBMPFD to include motor neuron degeneration, suggest that VCP mutations may account for ～1%-2% of familial ALS, and provide evidence directly implicating defects in the ubiquitination/protein degradation pathway in motor neuron degeneration.     

Structural insight into mutations at 155 position of valosin containing protein (VCP) linked to inclusion body myopathy with Paget disease of bone and frontotemporal Dementia

Mutations in Valosin-containing protein (VCP) have been implicated in the pathology linked to inclusion body myopathy, paget disease of bone and frontotemporal dementia (IBMPFD). VCP is an essential component of AAA-ATPase superfamily involved in various cellular functions. Advanced In-silico analysis was performed using prediction based servers to determine the most deleterious mutation. Molecular dynamics simulation was used to study the protein dynamics at atomic level. Molecular docking was used to study the effect of mutation on ATP/ADP transition in the kinase domain. This ATPase of 806 amino acids has four domains: N-terminal domain, C-terminal domain and two ATPase domains D1 and D2 and each of these domains have a distinct role in its functioning. The mutations in VCP protein are distributed among regions known as hotspots, one such hotspot is codon 155. Three missense mutations reported in this hotspot are R155C, R155H and R155P. Potentiality of the deleteriousness calculated using server based prediction models reveal R155C mutation to be the most deleterious. The atomic insight into the effect of mutation by molecular dynamics simulation revealed major conformational changes in R155C variants ATP binding site in D1 domain. The nucleotide-binding mode at the catalytic pocket of VCP and its three variants at codon 155 showed change in the structure, which affects the ATP–ADP transition kinetics in all the three variants.     

Specific Inhibition of p97/VCP ATPase and Kinetic Analysis Demonstrate Interaction between D1 and D2 ATPase Domains

The p97 AAA (ATPase associated with diverse cellular activities), also called VCP (valosin-containing protein), is an important therapeutic target for cancer and neurodegenerative diseases. p97 forms a hexamer composed of two AAA domains (D1 and D2) that form two stacked rings and an N-terminal domain that binds numerous cofactor proteins. The interplay between the three domains in p97 is complex, and a deeper biochemical understanding is needed in order to design selective p97 inhibitors as therapeutic agents. It is clear that the D2 ATPase domain hydrolyzes ATP in vitro, but whether D1 contributes to ATPase activity is controversial. Here, we use Walker A and B mutants to demonstrate that D1 is capable of hydrolyzing ATP and show for the first time that nucleotide binding in the D2 domain increases the catalytic efficiency (k_cat/K_m) of D1 ATP hydrolysis 280-fold, by increasing k_cat 7-fold and decreasing K_m about 40-fold. We further show that an ND1 construct lacking D2 but including the linker between D1 and D2 is catalytically active, resolving a conflict in the literature. Applying enzymatic observations to small-molecule inhibitors, we show that four p97 inhibitors (DBeQ, ML240, ML241, and NMS-873) have differential responses to Walker A and B mutations, to disease-causing IBMPFD mutations, and to the presence of the N domain binding cofactor protein p47. These differential effects provide the first evidence that p97 cofactors and disease mutations can alter p97 inhibitor potency and suggest the possibility of developing context-dependent inhibitors of p97.     

Capturing VCP: another molecular piece in the ALS jigsaw puzzle

TDP-43 mislocalization and aggregation are implicated in the pathogenesis of ALS and FTLD-U. Valosin containing protein (VCP) mutations also lead to TDP-43 deposition, resulting in Inclusion Body Myopathy, Paget disease, and Frontotemporal Dementia (IBMPFD). In this issue of Neuron, Johnson et?al. used whole-exome capture to identify VCP mutations in familial ALS. This extends the VCP phenotype to include motor neuron degeneration and provides another molecular tool to explore neurodegeneration disease mechanisms underlying the TDP-43 proteinopathies.     

Conformational changes of the multifunction p97 AAA ATPase during its ATPase cycle

p97 (also called VCP), a member of the AAA ATPase family, is involved in several cellular processes, including membrane fusion and extraction of proteins from the endoplasmic reticulum for cytoplasmic degradation. We have studied the conformational changes that p97 undergoes during the ATPase cycle by cryo-EM and single-particle analysis. Three-dimensional maps show that the two AAA domains, D1 and D2, as well as the N-domains, experience conformational changes during ATP binding, ATP hydrolysis, P(i) release and ADP release. The N-domain is flexible in most nucleotide states except after ATP hydrolysis. The rings formed by D1 and D2 rotate with respect to each other, and the size of their axial openings fluctuates. Taken together, our results depict the movements that this and possibly other AAA ATPases can undergo during an ATPase cycle.     

Structural and functional deviations in disease-associated p97 mutants

Missense mutations that occur at the interface between two functional domains in the AAA protein p97 lead to suboptimal performance in its enzymatic activity and impaired intracellular functions, causing human disorders such as inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia (IBMPFD). Much progress has been made in characterizing these mutants at cellular, sub-cellular and molecular levels, gaining a substantial understanding of the involvement of p97 in various cellular pathways. At the tissue level, patient biopsies revealed co-localization of p97 with pathologic proteineous inclusions and rimmed vacuoles, which can be reproduced in various cellular and animal models of IBMPFD. At the subcellular level, alterations in p97's ability to bind various adaptor proteins have been demonstrated for some but not all binding partners. Biochemical and biophysical characterizations of pathogenic p97 revealed altered nucleotide binding properties in the D1-domains compared to the wild type. Structural studies showed that mutant p97 are capable of undergoing a uniform transition in the N-domain from a Down- to an Up-conformation in the presence of ATPγS, while in the wild-type p97, this conformational change can only be demonstrated in solutions but not in crystals. These structural and biochemical analyses of IBMPFD mutants shed new light into the mechanism of p97 function.     

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

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 Mg²⁺·ATPγS, 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 ATPγS‐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.     

Neuronal remodeling and apoptosis require VCP-dependent degradation of the apoptosis inhibitor DIAP1

The regulated degeneration of axons or dendrites (pruning) and neuronal apoptosis are widely used during development to determine the specificity of neuronal connections. Pruning and apoptosis often share similar mechanisms; for example, developmental dendrite pruning of Drosophila class IV dendritic arborization (da) neurons is induced by local caspase activation triggered by ubiquitin-mediated degradation of the caspase inhibitor DIAP1. Here, we examined the function of Valosin-containing protein (VCP), a ubiquitin-selective AAA chaperone involved in endoplasmic reticulum-associated degradation, autophagy and neurodegenerative disease, in Drosophila da neurons. Strong VCP inhibition is cell lethal, but milder inhibition interferes with dendrite pruning and developmental apoptosis. These defects are associated with impaired caspase activation and high DIAP1 levels. In cultured cells, VCP binds to DIAP1 in a ubiquitin- and BIR domain-dependent manner and facilitates its degradation. Our results establish a new link between ubiquitin, dendrite pruning and the apoptosis machinery.     

Multifunctional roles of the conserved Arg residues in the second region of homology of p97/valosin-containing protein

The 97-kDa molecular chaperone valosin-containing protein (VCP) belongs to a highly conserved AAA family and forms a hexameric structure that is essential for its biological functions. The AAA domain contains highly conserved motifs, the Walker A, Walker B, and the second region of homology (SRH). Although Walker A and B motifs mediate ATP binding and hydrolysis, respectively, the function of the SRH in VCP is not clear. We examined the significance of the SRH in VCP, especially the conserved Arg(359) and Arg(362) in the first AAA domain, D1 and Arg(635) and Arg(638) in the second AAA domain, D2. We show that Arg(359) and Arg(362) in D1 are critical for maintaining the hexameric structure and the ability to bind the polyubiquitin chains. Although the rest of the tested SRH mutants retain the hexameric structure, all of them exhibit severely reduced ATPase activity. Tryptophan fluorescence analysis showed that all of the tested mutants can bind to ATP or ADP. Thus, the reduced ATPase activity likely results from the hampered communications among protomers during hydrolysis. Moreover, when the ATPase-defective mutant R635A or R638A is mixed with the Walker A mutant of D2, the ATPase activity is partially restored, suggesting that Arg(635) and Arg(638) can stimulate the ATPase activity of the neighboring protomer. Interestingly, mutation of Arg(359) and Arg(362) uncouples the inhibitory effect of p47, a VCP co-factor, on the ATPase activity of VCP. Therefore, the Arg residues allow D1 to take on a specific conformation that is required for substrate binding and co-factor communications. Taken together, these results demonstrate that the conserved Arg residues in the SRH of both D1 and D2 play critical roles in communicating the conformational changes required for ATP hydrolysis, and SRH in D1 also contributes to substrate binding and co-factor communications.