RecA-like motor ATPases--lessons from structures

A large class of ATPases contains a RecA-like structural domain and uses the energy of nucleotide binding and hydrolysis to perform mechanical work, for example, to move polypeptides or nucleic acids. These ATPases include helicases, ABC transporters, clamp loaders, and proteases. The functional units of the ATPases contain different numbers of RecA-like domains, but the nucleotide is always bound at the interface between two adjacent RecA-like folds and the two domains move relative to one another during the ATPase cycle. The structures determined for different RecA-like motor ATPases begin to reveal how they move macromolecules.     

Structure of the ABC ATPase domain of human TAP1, the transporter associated with antigen processing

The transporter associated with antigen processing (TAP) is an ABC transporter formed of two subunits, TAP1 and TAP2, each of which has an N-terminal membrane-spanning domain and a C-terminal ABC ATPase domain. We report the structure of the C-terminal ABC ATPase domain of TAP1 (cTAP1) bound to ADP. cTAP1 forms an L-shaped molecule with two domains, a RecA-like domain and a small alpha-helical domain. The diphosphate group of ADP interacts with the P-loop as expected. Residues thought to be involved in gamma-phosphate binding and hydrolysis show flexibility in the ADP-bound state as evidenced by their high B-factors. Comparisons of cTAP1 with other ABC ATPases from the ABC transporter family as well as ABC ATPases involved in DNA maintenance and repair reveal key regions and residues specific to each family. Three ATPase subfamilies are identified which have distinct adenosine recognition motifs, as well as distinct subdomains that may be specific to the different functions of each subfamily. Differences between TAP1 and TAP2 in the nucleotide-binding site may be related to the observed asymmetry during peptide transport.     

Single-molecule fluorescence resonance energy transfer techniques on rotary ATP synthases

Conformational changes of proteins can be monitored in real time by fluorescence resonance energy transfer (FRET). Two different fluorophores have to be attached to those protein domains which move during function. Distance fluctuations between the fluorophores are measured by relative fluorescence intensity changes or fluorescence lifetime changes. The rotary mechanics of the two motors of F(o)F(1)-ATP synthase have been studied in vitro by single-molecule FRET. The results are summarized and perspectives for other transport ATPases are discussed.     

Rad50/SMC proteins and ABC transporters: unifying concepts from high-resolution structures

ATP-binding cassette (ABC)-type ATPases are chemo-mechanical engines for diverse biological pathways. ABC ATPase domains act not only in ABC transporters but also in DNA mismatch, nucleotide excision and double-strand break repair enzymes, as well as in chromosome segregation. Atomic-resolution crystal structures suggest molecular mechanisms for ABC ATPases and reveal surprisingly significant mechanistic and architectural conservation. This emerging unified structural biochemistry provides general medical and biological insights into how ABC proteins function as chemo-mechanical devices. ATP binding by the signature and Q-loop motifs drives the conformations of substrate-specific domains to accomplish diverse functions in transmembrane transport and DNA repair.     

Functional modules of KdpB, the catalytic subunit of the Kdp-ATPase from Escherichia coli

The large cytoplasmic domain (H4H5) of KdpB of the KdpFABC complex (P-type ATPase) from Escherichia coli consists of two separate modules, the phosphorylation domain (KdpBP) and the nucleotide binding domain (KdpBN). The H4H5 and the KdpBN domains were purified as soluble 10His-tagged fusion proteins. Both proteins exhibit a mainly alpha-helical secondary structure as judged by CD spectroscopy. Fluorescein 5-isothiocyanate (FITC) labeling studies revealed that both proteins form a proper nucleotide binding site. Adenosine nucleotides protect the H4H5 loop but not KdpBN against FITC modification. Trinitrophenyl (TNP)-nucleotide binding studies revealed that both H4H5 and KdpBN bind nucleotides with high affinity. Furthermore, the H4H5 loop was still able to hydrolyze ATP, as well as p-nitrophenyl phosphate (pNPP). These results lend support to the notion that the separately synthesized H4H5 and KdpBN domains retain their native structure and that they reveal properties of both P2-type ATPases (e.g., Na(+),K(+)-ATPase and Ca(2+)-ATPase) and P1b-type ATPases (e.g., heavy metal transporting ATPases). Furthermore, this report also emphasizes the unique position of the Kdp-ATPase within the P-type ATPase family.     

Structural and functional insights into the human Upf1 helicase core

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance pathway that recognizes and degrades aberrant mRNAs containing premature stop codons. A critical protein in NMD is Upf1p, which belongs to the helicase super family 1 (SF1), and is thought to utilize the energy of ATP hydrolysis to promote transitions in the structure of RNA or RNA-protein complexes. The crystal structure of the catalytic core of human Upf1p determined in three states (phosphate-, AMPPNP- and ADP-bound forms) reveals an overall structure containing two RecA-like domains with two additional domains protruding from the N-terminal RecA-like domain. Structural comparison combined with mutational analysis identifies a likely single-stranded RNA (ssRNA)-binding channel, and a cycle of conformational change coupled to ATP binding and hydrolysis. These conformational changes alter the likely ssRNA-binding channel in a manner that can explain how ATP binding destabilizes ssRNA binding to Upf1p.     

Improved structures of full-length p97, an AAA ATPase: implications for mechanisms of nucleotide-dependent conformational change

The ATPases associated with various cellular activities (AAA) protein p97 has been implicated in a variety of cellular processes, including endoplasmic reticulum-associated degradation and homotypic membrane fusion. p97 belongs to a subgroup of AAA proteins that contains two nucleotide binding domains, D1 and D2. We determined the crystal structure of D2 at 3.0 A resolution. This model enabled rerefinement of full-length p97 in different nucleotide states against previously reported low-resolution diffraction data to significantly improved R values and Ramachandran statistics. Although the overall fold remained similar, there are significant improvements, especially around the D2 nucleotide binding site. The rerefinement illustrates the importance of knowledge of high-resolution structures of fragments covering most of the whole molecule. The structures suggest that nucleotide hydrolysis is transformed into larger conformational changes by pushing of one D2 domain by its neighbor in the hexamer, and transmission of nucleotide-state information through the D1-D2 linker to displace the N-terminal, effector binding domain.     

Conformational variability of the GTPase domain of the signal recognition particle receptor FtsY

The prokaryotic signal recognition particle Ffh and its receptor FtsY allow targeting of proteins into or across the plasma membrane. The targeting process is GTP dependent and the two proteins constitute a distinct GTPase family. The receptor FtsY is composed of A and NG domains where the NG's GTPase domain plays a critical role in the targeting process. In this study, we describe two X-ray structures determined independently of each other of the NG domain of FtsY from Mycoplasma mycoides (MmFtsY). The two structures are markedly different in three of the nucleotide-binding segments, GI (P-loop), GII, and GIII, making only one of the structures compatible with nucleotide binding. Interestingly, the two distinct conformations of the nucleotide-binding segments of MmFtsY are similar to the apo- and ADP-loaded forms of certain ATPases. The structure of the extended interface between the A and NG domains of MmFtsY provides new insights into the role of the A domain for phospholipid interaction.     

Complementary DNA cloning of a protein highly homologous to mammalian sarcoplasmic reticulum Ca-ATPase from the crustacean Artemia

Complementary DNA clones coding for an Artemia ATPase have been isolated using an oligonucleotide probe for a region highly conserved between P-type ATPases. The nucleotide sequence of three overlapping clones, 3309 base-pairs, has been established. This sequence includes 78 nucleotides of 5' untranslated sequence, an open reading frame of 3009 nucleotides and 222 nucleotides of 3' untranslated sequences. The amino acid sequence predicted for the coding region is 71% similar to that of slow and fast twitch rabbit muscle sarcoplasmic reticulum Ca-ATPases. The homology is specially high in some regions of the protein that include the previously described regions that are similar between all known P-type ATPases, as well as transmembrane domains and intra- and extracellular domains adjacent to the membrane that are not conserved in P-type ATPases but have been proposed to be involved in calcium binding and transport in rabbit sarcoplasmic reticulum Ca-ATPases. Probes of this likely sarcoplasmic reticulum Ca-ATPase hybridize to two mRNAs of 5200 and 4500 bases. Although both mRNAs are already present in cryptobiotic embryos, the levels of the 5200 base mRNA decrease after development is reassumed, being undetectable after hatching of the nauplii. The levels of the 4500 base mRNA increase during development; maximal levels are reached by ten hours and are maintained at later stages of development.     

Structural frameworks for considering microbial protein- and nucleic acid-dependent motor ATPases

Many fundamental cellular processes depend on enzymes that utilize chemical energy to catalyse unfavourable reactions. Certain classes of ATPases provide a particularly vivid example of the process of energy conversion, employing cycles of nucleotide turnover to move and/or rearrange biological polymers such as proteins and nucleic acids. Four well-characterized classes of ATP-dependent protein/nucleic acid translocases and remodelling factors are found in all three domains of life (bacteria, archaea and eukarya): additional strand catalytic 'E' (ASCE) P-loop NTPases, GHL proteins, actin-fold enzymes and chaperonins. These unrelated protein superfamilies have each evolved the ability to couple ATP binding and hydrolysis to the generation of motion and force along or within their substrates. The past several years have witnessed the emergence of a wealth of structural data that help explain how such molecular engines link nucleotide turnover to conformational change. In this review, we highlight several recent advances to illustrate some of the mechanisms by which each family of ATP-dependent motors facilitates the rearrangement and movement of proteins, protein complexes and nucleic acids.     

Structural divergence between the two subgroups of P5 ATPases

Evolution of P5 type ATPases marks the origin of eukaryotes but still they remain the least characterized pumps in the superfamily of P-type ATPases. Phylogenetic analysis of available sequences suggests that P5 ATPases should be divided into at least two subgroups, P5A and P5B. P5A ATPases have been identified in the endoplasmic reticulum and seem to have basic functions in protein maturation and secretion. P5B ATPases localize to vacuolar/lysosomal or apical membranes and in animals play a role in hereditary neuronal diseases. Here we have used a bioinformatical approach to identify differences in the primary sequences between the two subgroups. P5A and P5B ATPases appear have a very different membrane topology from other P-type ATPases with two and one, respectively, additional transmembrane segments inserted in the N-terminal end. Based on conservation of residues in the transmembrane region, the two P5 subgroups most likely have different substrate specificities although these cannot be predicted from their sequences. Furthermore, sequence differences between P5A and P5B ATPases are identified in the catalytic domains that could influence key kinetic properties differentially. Together these findings indicate that P5A and P5B ATPases are structurally and functionally different.     

The RIG-I ATPase domain structure reveals insights into ATP-dependent antiviral signalling

RIG-I detects cytosolic viral dsRNA with 5' triphosphates (5'-ppp-dsRNA), thereby initiating an antiviral innate immune response. Here we report the crystal structure of superfamily 2 (SF2) ATPase domain of RIG-I in complex with a nucleotide analogue. RIG-I SF2 comprises two RecA-like domains 1A and 2A and a helical insertion domain 2B, which together form a 'C'-shaped structure. Domains 1A and 2A are maintained in a 'signal-off' state with an inactive ATP hydrolysis site by an intriguing helical arm. By mutational analysis, we show surface motifs that are critical for dsRNA-stimulated ATPase activity, indicating that dsRNA induces a structural movement that brings domains 1A and 2A/B together to form an active ATPase site. The structure also indicates that the regulatory domain is close to the end of the helical arm, where it is well positioned to recruit 5'-ppp-dsRNA to the SF2 domain. Overall, our results indicate that the activation of RIG-I occurs through an RNA- and ATP-driven structural switch in the SF2 domain.     

Nucleotide dependent motion and mechanism of action of p97/VCP

The AAA (ATPases associated with a variety of cellular activities) family of proteins bind, hydrolyze, and release ATP to effect conformational changes, assembly, or disassembly upon their binding partners and substrate molecules. One of the members of this family, the hexameric p97/valosin-containing protein p97/VCP, is essential for the dislocation of misfolded membrane proteins from the endoplasmic reticulum. Here, we observe large motions and dynamic changes of p97/VCP as it proceeds through the ATP hydrolysis cycle. The analysis is based on crystal structures of four representative ATP hydrolysis states: APO, AMP-PNP, hydrolysis transition state ADP x AlF3, and ADP bound. Two of the structures presented herein, ADP and AMP-PNP bound, are new structures, and the ADP x AlF3 structure was re-refined to higher resolution. The largest motions occur at two stages during the hydrolysis cycle: after, but not upon, nucleotide binding and then following nucleotide release. The motions occur primarily in the D2 domain, the D1 alpha-helical domain, and the N-terminal domain, relative to the relatively stationary and invariant D1alpha/beta domain. In addition to the motions, we observed a transition from a rigid state to a flexible state upon loss of the gamma-phosphate group, and a further increase in flexibility within the D2 domains upon nucleotide release. The domains within each protomer of the hexameric p97/VCP deviate from strict 6-fold symmetry, with the more flexible ADP state exhibiting greater asymmetry compared to the relatively rigid ADP x AlF3 state, suggesting a mechanism of action in which hydrolysis and conformational changes move about the hexamer in a processive fashion.     

Changing nucleotide specificity of the DEAD-box helicase Hera abrogates communication between the Q-motif and the P-loop

DEAD-box proteins disrupt or remodel RNA and protein/RNA complexes at the expense of ATP. The catalytic core is composed of two flexibly connected RecA-like domains. The N-terminal domain contains most of the motifs involved in nucleotide binding and serves as a minimalistic model for helicase/nucleotide interactions. A single conserved glutamine in the so-called Q-motif has been suggested as a conformational sensor for the nucleotide state. To reprogram the Thermus thermophilus RNA helicase Hera for use of oxo-ATP instead of ATP and to investigate the sensor function of the Q-motif, we analyzed helicase activity of Hera Q28E. Crystal structures of the Hera N-terminal domain Q28E mutant (TthDEAD_Q28E) in apo- and ligand-bound forms show that Q28E does change specificity from adenine to 8-oxoadenine. However, significant structural changes accompany the Q28E mutation, which prevent the P-loop from adopting its catalytically active conformation and explain the lack of helicase activity of Hera_Q28E with either ATP or 8-oxo-ATP as energy sources. 8-Oxo-adenosine, 8-oxo-AMP, and 8-oxo-ADP weakly bind to TthDEAD_Q28E but in non-canonical modes. These results indicate that the Q-motif not only senses the nucleotide state of the helicase but could also stabilize a catalytically competent conformation of the P-loop and other helicase signature motifs.     

F-type or V-type? The chimeric nature of the archaebacterial ATP synthase.

Archaebacterial plasma membranes contain an ATPase acting in vivo as a delta mu H(+)-driven ATP synthase. While functional features and their general structural design are resembling F-type ATPases, primary sequences of the two large polypeptides from the catalytic part are closely related to V-type ATPases from eucaryotic vacuolar membranes. The chimeric nature of archaebacterial ATPase from Sulfolobus was investigated in terms of nucleotide interactions and related to specific sequence parameters in a comparison to well known F- and V-type ATPases. The study disclosed a general difference of F- and V-type ATPases at one class of the nucleotide binding sites.     

Crystal structures of the pilus retraction motor PilT suggest large domain movements and subunit cooperation drive motility

PilT is a hexameric ATPase required for bacterial type IV pilus retraction and surface motility. Crystal structures of ADP- and ATP-bound Aquifex aeolicus PilT at 2.8 and 3.2 A resolution show N-terminal PAS-like and C-terminal RecA-like ATPase domains followed by a set of short C-terminal helices. The hexamer is formed by extensive polar subunit interactions between the ATPase core of one monomer and the N-terminal domain of the next. An additional structure captures a nonsymmetric PilT hexamer in which approach of invariant arginines from two subunits to the bound nucleotide forms an enzymatically competent active site. A panel of pilT mutations highlights the importance of the arginines, the PAS-like domain, the polar subunit interface, and the C-terminal helices for retraction. We present a model for ATP binding leading to dramatic PilT domain motions, engagement of the arginine wire, and subunit communication in this hexameric motor. Our conclusions apply to the entire type II/IV secretion ATPase family.     

Cloning and Characterization of an ATPase Gene from Pneumocystis carinii which Closely Resembles Fungal H+ ATPases

ABSTRACT. A gene encoding a P-type cation translocating ATPase was cloned from a genomic library of rat-derived Pneumocystis carinii. The nucleotide sequence of the gene contains a 2781 base-pair open reading frame that is predicted to encode a 101, 401 dalton protein composed of 927 amino acids. The P. carinii ATPase protein (pcal) is 69–75% identical when compared with eight proton pumps from six fungal species. The Pneumocystis ATPase is less than 34% identical to ATPase proteins from protozoans, vertebrates or the Ca⁺⁺ ATPases of yeast. The P. carinii ATPase contains 115 of 121 residues previously identified as characteristic of H⁺ ATPases. Alignment of the Pneumocystis and fungal proton pumps reveals five homologous domains specific for fungal H⁺ ATPases.     

An Exceptional Variability in the Motor of Archaeal A1A0 ATPases: From Multimeric to Monomeric Rotors Comprising 6–13 Ion Binding Sites

The motor domain of A1A0 ATPases is composed of only two subunits, the stator subunit I and the rotor subunit c. Recent studies on the molecular biology of the A0 domains revealed the surprising finding that the gene encoding subunit c underwent several multiplication events leading to rotor subunits comprising 2, 3, or even 13 hairpin domains suggesting multimeric in different stoichiometry as well as monomeric rotors. The number of ion translocating groups per rotor ranges from 13 to 6. Furthermore, as deduced from the gene sequences H(+)-as well as Na(+)-driven rotors are found in archaea. Features previously thought to be distinctive for A0, F0 or V0 are all found in A0 suggesting that the differences encountered in the three classes of ATPases today emerged already very early in evolution. The extraordinary features and exceptional structural and functional variability in the rotor of A1A0 ATPases may have arisen as an adaptation to different cellular needs and the extreme physicochemical conditions in the early history of life.