Biology subjects

Jeruzalmi, D.

Publications and source records attributed to Jeruzalmi, D..

4 recordsLinked to original sources

bioRxiv2024

DnaB and DciA: Mechanisms of Helicase Loading and Translocation on ssDNA

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/622779v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@b0641dorg.highwire.dtl.DTLVardef@7caf59org.highwire.dtl.DTLVardef@1dc0fccorg.highwire.dtl.DTLVardef@913ce2_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO The Vibrio cholerae (Vc) DnaB replicative helicase structure bound to single-stranded (ss) DNA is depicted in the ribbon (top left) and sphere-cylinder representation (top right). In the bottom center is a native mass spectrum showing Vc DnaB helicase loading onto single-stranded DNA (ssDNA). C_FIG Replicative helicases are assembled on chromosomes by helicase loaders before initiation of DNA replication. Here, we investigate mechanisms used by the bacterial Vibrio cholerae (Vc) DnaB replicative helicase and the DciA helicase loader. Structural analysis of the ATP{gamma}S form of the VcDnaB-ssDNA complex reveals a configuration distinct from that seen with GDP*AlF4. With ATP{gamma}S, the amino-terminal (NTD) tier, previously found as an open spiral in the GDP*AlF4 complex, adopts a closed planar arrangement. Further, the DnaB subunit at the top of the carboxy-terminal spiral (CTD) tier is displaced by [~]25 [A] between the two forms. We suggest that remodeling the NTD layer between closed planar and open spiral configurations and migration of two distinct CTDs to the top of the DnaB spiral, repeated three times, mediates hand-over-hand translocation. Biochemical analysis suggests that VcDciA leverages its Lasso domain to contact DnaB near its Docking-Linker-Helix interface. Up to three copies of VcDciA bind to VcDnaB and suppress its ATPase activity during loading onto physiological DNA substrates. Our data suggest that DciA loads DnaB onto DNA using the ring-opening mechanism.

biochemistry↗

bioRxiv2022

An Autoinhibited Conformation of the DnaB-Replicative Helicase phage Lambda P Loader Complex

Replicative helicases require loader proteins for assembly at the origins of DNA replication. Multiple copies of the bacteriophage {lambda}P (P) loader bind to and load the E. coli DnaB (B) replicative helicase on replication-origin-derived single-stranded DNA. We find that the E. coli DnaB*{lambda}P complex exists in two forms: B6P5 and B6P6. In the 2.66 [A] cryo-EM model of B6P5, five copies of the {lambda}P loader assemble into a crown-like shape that tightly grips DnaB. In this complex, closed planar DnaB is reconfigured into an open spiral with a sufficiently sized breach to permit ssDNA to enter an internal chamber. The transition to the open spiral involves {lambda}P-mediated changes to the Docking Helix (DH)-Linker Helix (LH) interface. The loader directly stabilizes the open spiral. Unexpectedly, one {lambda}P chain in B6P5 is bound across the breach, precluding entry of replication-origin-derived ssDNA into DnaBs central chamber. We suggest that the B6P6 complex is an early intermediate in the helicase activation pathway wherein neither the DnaB helicase nor the {lambda}P loader has attained its final form. DnaB in this complex adopts a partially open planar configuration, termed ajar planar. The partially ordered {lambda}P loader assembly features a much looser interaction with DnaB. The ssDNA and ATP sites in both complexes are in a configuration ill-suited for binding or hydrolysis. Our work specifies the conformational changes required for the intermediate B6P6 to transition to B6P5 on the pathway to recruitment by the initiator protein complex to the replication origin. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/522210v4_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1c44809org.highwire.dtl.DTLVardef@1554deeorg.highwire.dtl.DTLVardef@16f6df9org.highwire.dtl.DTLVardef@9d12dd_HPS_FORMAT_FIGEXP M_FIG C_FIG The DnaB helicase loading pathway at the phage {lambda} replication origin populates two intermediate states, distinguished by the number of {lambda}P loaders present. The DnaB ring in the B6P6 complex is planar and partially open. Although it binds six copies of the {lambda}P loader, the ajar planar state of DnaB yields inchoate interactions with the loader. During the maturation of the complex, the planar state of DnaB is reconfigured into an open spiral in the B6P5 complex, which the pentameric {lambda}P ensemble now grips tightly. This transition required the eviction of one copy of the loader. Although the breach in the DnaB open spiral is sufficiently sized for entry of ssDNA into the internal chamber, the disposition of one {lambda}P chain loader across the single breached interface in DnaB effectively blocks the path to physiological replication origin-derived ssDNA. DnaB is depicted in white/gray ribbon format under a transparent surface. The {lambda}P chains are colored orange and shades of blue. The pink cylinder represents the expected path of ssDNA through DnaB.

biochemistry↗

bioRxiv2022

Structural Basis for the Calmodulin-Mediated Activation of eEF-2K

Translation is a highly energy consumptive process1 tightly regulated for optimal protein quality2 and adaptation to energy and nutrient availability. A key facilitator of this process is the -kinase eEF-2K that specifically phosphorylates the GTP-dependent translocase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis3,4. eEF-2K activation requires calmodulin binding and auto-phosphorylation at the primary stimulatory site, T348. Biochemical studies have predicted that calmodulin activates eEF-2K through a unique allosteric process5 mechanistically distinct from other calmodulin-dependent kinases6. Here we resolve the atomic details of this mechanism through a 2.3 [A] crystal structure of the heterodimeric complex of calmodulin with the functional core of eEF-2K (eEF-2KTR). This structure, which represents the activated T348-phosphorylated state of eEF-2KTR, highlights how through an intimate association with the calmodulin C-lobe, the kinase creates a "spine" that extends from its N-terminal calmodulin-targeting motif through a conserved regulatory element to its active site. Modification of key spine residues has deleterious functional consequences.

biochemistry↗

bioRxiv2017

Routine Single Particle CryoEM Sample and Grid Characterization by Tomography

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are freely floating away from the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1,000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

biophysics↗