It has been proposed that the reaction cycle of ATP binding cassette (ABC) transporters is driven by dimerization of their ABC motor domains upon binding ATP at their mutual interface. However, no such ATP sandwich complex has been observed for an ABC from an ABC transporter. In this paper, we report the crystal structure of a stable dimer formed by the E171Q mutant of the MJ0796 ABC, which is hydrolytically inactive due to mutation of the catalytic base. The structure shows a symmetrical dimer in which two ATP molecules are each sandwiched between the Walker A motif in one subunit and the LSGGQ signature motif in the other subunit. These results establish the stereochemical basis of the power stroke of ABC transporter pumps.
ABC-F proteins have evaded functional characterization even though they comprise one of the most widely distributed branches of the ATP-binding cassette (ABC) superfamily. Herein, we demonstrate that YjjK, the most prevalent eubacterial ABC-F protein, gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. Accordingly, we rename this protein Energy-dependent Translational Throttle A (EttA). We determined the crystal structure of Escherichia coli EttA and used it to design mutants for biochemical studies, including enzymological assays of the initial steps of protein synthesis. These studies suggest that EttA may regulate protein synthesis in energy-depleted cells, which have a low ATP/ADP ratio. Consistent with this inference, ΔettA cells exhibit a severe fitness defect in long-term stationary phase. These studies demonstrate that an ABC-F protein regulates protein synthesis via a novel mechanism sensitive to cellular energy status.
Chlorella virus DNA ligase, the smallest eukaryotic ligase known, has pluripotent biological activity and an intrinsic nick-sensing function, despite having none of the accessory domains found in cellular ligases. A 2.3-A crystal structure of the Chlorella virus ligase-AMP intermediate bound to duplex DNA containing a 3'-OH-5'-PO4 nick reveals a new mode of DNA envelopment, in which a short surface loop emanating from the OB domain forms a beta-hairpin 'latch' that inserts into the DNA major groove flanking the nick. A network of interactions with the 3'-OH and 5'-PO4 termini in the active site illuminates the DNA adenylylation mechanism and the crucial roles of AMP in nick sensing and catalysis. Addition of a divalent cation triggered nick sealing in crystallo, establishing that the nick complex is a bona fide intermediate in the DNA repair pathway.
BtuF is the periplasmic binding protein (PBP) for the vitamin B12 transporter BtuCD, a member of the ATPbinding cassette (ABC) transporter superfamily of transmembrane pumps. We have determined crystal structures of Escherichia coli BtuF in the apo state at 3.0 Å resolution and with vitamin B12 bound at 2.0 Å resolution. The structure of BtuF is similar to that of the FhuD and TroA PBPs and is composed of two ␣/ domains linked by a rigid ␣-helix. B12 is bound in the "base-on" or vitamin conformation in a wide acidic cleft located between these domains. The C-terminal domain shares structural homology to a B12-binding domain found in a variety of enzymes. The same surface of this domain interacts with opposite surfaces of B12 when comparing ligand-bound structures of BtuF and the homologous enzymes, a change that is probably caused by the obstruction of the face that typically interacts with this domain by the base-on conformation of vitamin B12 bound to BtuF. There is no apparent pseudo-symmetry in the surface properties of the BtuF domains flanking its B12 binding site even though the presumed transport site in the previously reported crystal structure of BtuCD is located in an intersubunit interface with 2-fold symmetry. Unwinding of an ␣-helix in the C-terminal domain of BtuF appears to be part of conformational change involving a general increase in the mobility of this domain in the apo structure compared with the B12-bound structure. As this helix is located on the surface likely to interact with BtuC, unwinding of the helix upon binding to BtuC could play a role in triggering release of B12 into the transport cavity. Furthermore, the high mobility of this domain in free BtuF could provide an entropic driving force for the subsequent release of BtuF required to complete the transport cycle.
The SecA ATPase drives the processive translocation of the N terminus of secreted proteins through the cytoplasmic membrane in eubacteria via cycles of binding and release from the SecYEG translocon coupled to ATP turnover. SecA forms a physiological dimer with a dissociation constant that has previously been shown to vary with temperature and ionic strength. We now present data showing that the oligomeric state of SecA in solution is altered by ligands that it interacts with during protein translocation. Analytical ultracentrifugation, chemical cross-linking, and fluorescence anisotropy measurements show that the physiological dimer of SecA is monomerized by long-chain phospholipid analogues. Addition of wild-type but not mutant signal sequence peptide to these SecA monomers redimerizes the protein. Physiological dimers of SecA do not change their oligomeric state when they bind signal sequence peptide in the compact, low temperature conformational state but polymerize when they bind the peptide in the domain-dissociated, high-temperature conformational state that interacts with SecYEG. This last result shows that, at least under some conditions, signal peptide interactions drive formation of new intermolecular contacts distinct from those stabilizing the physiological dimer. The observations that signal peptides promote conformationally specific oligomerization of SecA while phospholipids promote subunit dissociation suggest that the oligomeric state of SecA could change dynamically during the protein translocation reaction. Cycles of SecA subunit recruitment and dissociation could potentially be employed to achieve processivity in polypeptide transport.
The DNA ligase D (LigD) 3′-phosphoesterase (PE) module is a conserved component of the bacterial nonhomologous end-joining (NHEJ) apparatus that performs 3′ end-healing reactions at DNA double-strand breaks. Here we report the 1.9 Å crystal structure of Pseudomonas aeruginosa PE, which reveals that PE exemplifies a unique class of DNA repair enzyme. PE has a distinctive fold in which an eight stranded β barrel with a hydrophobic interior supports a crescent-shaped hydrophilic active site on its outer surface. Six essential side chains coordinate manganese and a sulfate mimetic of the scissile phosphate. The PE active site and mechanism are unique vis à vis other end-healing enzymes. We find PE homologs in archaeal and eukaryal proteomes, signifying that PEs comprise a DNA repair superfamily.is the key agent of the bacterial nonhomologous end-joining (NHEJ) pathway of DNA doublestrand break (DSB) repair (1). LigD is a single polypeptide consisting of three autonomous catalytic domain modules: an ATP-dependent ligase (LIG), a polymerase (POL), and a 3′-phosphoesterase (PE). The POL domain incorporates dNMP/rNMPs at DSB ends and gaps prior to strand sealing by the LIG domain (2-6) and is responsible, in large part, for the mutagenic outcomes of bacterial NHEJ in vivo (7). The PE domain provides a 3′ end-healing function, whereby it cleans up "dirty" DSBs with 3′-phosphate ends (8). PE also trims short 3′-ribonucleotide tracts (produced by POL) to generate the 3′ monoribonucleotide ends that are the preferred substrates for sealing by bacterial NHEJ ligases (3,8). The biochemical properties and atomic structures of the LIG and POL domains highlighted their membership in the covalent nucleotidyltransferase and archaeal/ eukaryal primase-polymerase families respectively (5, 9, 10). By contrast, the PE domain appears to be sui generis.The properties of the PE domain elucidated initially for Pseudomonas LigD also apply to the PE modules of Agrobacterium and Mycobacterium LigD (8,11,12). Specifically, PE displays a distinctive manganese-dependent 3′-ribonuclease/3′-phosphatase activity, entailing two component steps: (i) the 3′-terminal nucleoside is removed to yield a primer strand with a ribonucleoside 3′-PO 4 terminus; (ii) the 3′-PO 4 is hydrolyzed to a 3′-OH (Fig. 1A). PE activity is acutely dependent on the presence and length of a 5′ single-strand tail on a duplex primer-template substrate, thus implicating PE in 3′ end repair at gaps or recessed DSBs. Structure probing of Pseudomonas PE in solution revealed an apparently disordered N-terminal 29-aa segment, punctuated by a cluster of trypsin-and chymotrypsin-sensitive sites (Fig. 1B), flanking a seemingly well folded (i.e., protease insensitive) C-terminal domain (13). Deletion of the protease-sensitive N-terminal peptide had no effect on the phosphodiesterase activity of PE, though monoesterase activity was reduced. Mutational analyses identified an ensemble of conserved side chain functional groups within the protease-resistant module that were essential for ph...
RNA triphosphatase catalyzes the first step in mRNA capping. The RNA triphosphatases of fungi and protozoa are structurally and mechanistically unrelated to the analogous mammalian enzyme, a situation that recommends RNA triphosphatase as an anti-infective target. Fungal and protozoan RNA triphosphatases belong to a family of metal-dependent phosphohydrolases exemplified by yeast Cet1. The Cet1 active site is unusually complex and located within a topologically closed hydrophilic b-barrel (the triphosphate tunnel). Here we probe the active site of Plasmodium falciparum RNA triphosphatase by targeted mutagenesis and thereby identify eight residues essential for catalysis. The functional data engender an improved structural alignment in which the Plasmodium counterparts of the Cet1 tunnel strands and active-site functional groups are located with confidence. We gain insight into the evolution of the Cet1-like triphosphatase family by noting that the heretofore unique tertiary structure and active site of Cet1 are recapitulated in recently deposited structures of proteins from Pyrococcus (PBD 1YEM) and Vibrio (PDB 2ACA). The latter proteins exemplify a CYTH domain found in CyaB-like adenylate cyclases and mammalian thiamine triphosphatase. We conclude that the tunnel fold first described for Cet1 is the prototype of a larger enzyme superfamily that includes the CYTH branch. This superfamily, which we name ''triphosphate tunnel metalloenzyme,'' is distributed widely among bacterial, archaeal, and eukaryal taxa. It is now clear that Cet1-like RNA triphosphatases did not arise de novo in unicellular eukarya in tandem with the emergence of caps as the defining feature of eukaryotic mRNA. They likely evolved by incremental changes in an ancestral tunnel enzyme that conferred specificity for RNA 59-end processing.
PDB Reference: Cas2, 3oq2.CRISPRs (clustered regularly interspaced short palindromic repeats) provide bacteria and archaea with RNA-guided acquired immunity to invasive DNAs. CRISPR-associated (Cas) proteins carry out the immune effector functions. Cas2 is a universal component of the CRISPR system. Here, a 1.35 Å resolution crystal structure of Cas2 from the bacterium Desulfovibrio vulgaris (DvuCas2) is reported. DvuCas2 is a homodimer, with each protomer consisting of an N-terminal ferredoxin fold (amino acids 1-78) to which is appended a C-terminal segment (amino acids 79-102) that includes a short 3 10 -helix and a fifth -strand. The 5 strands align with the 4 strands of the opposite protomers, resulting in two five-stranded antiparallel -sheets that form a sandwich at the dimer interface. The DvuCas2 dimer is stabilized by a distinctive network of hydrophilic cross-protomer side-chain interactions.
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