Determination of the atomic resolution structure of a DNA Polymerase I isolated from Rhodothermus marinus

DNA polymerase I employs a multistep mechanism for sorting correctly paired nucleotides from mismatches. We aim to characterize reaction intermediates during nucleotide selection to better understand how this class of enzymes achieves high DNA replication fidelity. DNA polymerase I from R. marinus contains an unusual and disruptive proline in the mobile O helix near the active site. To characterize this enzyme, the structure of the large (5´-to-3´ exo-deficient) fragment of the R. marinus DNA polymerase I (RF) was solved to 2.95 Å (R = 0.234) using multi-wavelength anomalous dispersion. Alignment with homologous Escherichia coli Klenow Fragment (KF) DNA polymerase I confirmed that the active sites of each structural domain were conserved. In order to study the polymerase activity in isolation, 3’-to-5’ exonuclease activity was eliminated using site-directed mutagenesis of an aspartic acid involved in binding DNA (D497A). Unexpectedly, mutation of an aspartic acid involved in binding a catalytic magnesium ion (D421A in RF) failed to abolish exonuclease activity despite its ability to do so in the KF (D424A). Future structural studies will include crystallization of the polymerase in both its binary and ternary complex to study the conformational changes associated with the process of nucleotide selection as well as examination of the exonuclease domain to characterize its unusual activity in this enzyme

Reaching the end of the line: Urinary tract infections

Urinary tract infections (UTIs) cause a substantial health care burden. UTIs (i) are most often caused by uropathogeni

Red blood cell invasion by Plasmodium vivax: Structural basis for DBP engagement of DARC

Plasmodium parasites use specialized ligands which bind to red blood cell (RBC) receptors during invasion. Defining the mechanism of receptor recognition is essential for the design of interventions against malaria. Here, we present the structural basis for Duffy antigen (DARC) engagement by P. vivax Duffy binding protein (DBP). We used NMR to map the core region of the DARC ectodomain contacted by the receptor binding domain of DBP (DBP-RII) and solved two distinct crystal structures of DBP-RII bound to this core region of DARC. Isothermal titration calorimetry studies show these structures are part of a multi-step binding pathway, and individual point mutations of residues contacting DARC result in a complete loss of RBC binding by DBP-RII. Two DBP-RII molecules sandwich either one or two DARC ectodomains, creating distinct heterotrimeric and heterotetrameric architectures. The DARC N-terminus forms an amphipathic helix upon DBP-RII binding. The studies reveal a receptor binding pocket in DBP and critical contacts in DARC, reveal novel targets for intervention, and suggest that targeting the critical DARC binding sites will lead to potent disruption of RBC engagement as complex assembly is dependent on DARC binding. These results allow for models to examine inter-species infection barriers, Plasmodium immune evasion mechanisms, P. knowlesi receptor-ligand specificity, and mechanisms of naturally acquired P. vivax immunity. The step-wise binding model identifies a possible mechanism by which signaling pathways could be activated during invasion. It is anticipated that the structural basis of DBP host-cell engagement will enable development of rational therapeutics targeting this interaction

Heterotrimer interface residues determined by PDBePISA [47]: All residues in the interface are listed sequentially and do not indicate interacting pairs.

<p>Heterotrimer interface residues determined by PDBePISA <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003869#ppat.1003869-Krissinel1" target="_blank">[47]</a>: All residues in the interface are listed sequentially and do not indicate interacting pairs.</p

Binding interfaces of the DBP-RII∶DARC heterotrimer.

<p>(<b>A</b>) Global view of the DBP-RII∶DARC heterotrimer, showing (<b>B</b>) DARC monomer A interactions and (<b>C</b>) the DBP-RII homodimeric interface. DARC monomer A is in purple, DBP-RII monomer 1 is in green and DBP-RII monomer 2 is in yellow. Residue numbers are labeled and DARC residue labels are underlined.</p

Residues 14–43 of DARC contain the minimal binding region.

<p>¹H-¹⁵N-TROSY spectra of unbound DARC 1–60 (black) overlaid on ¹H-¹⁵N-TROSY spectra of DARC 1–60 in the presence of excess unlabelled DBP-RII (red). Sequence assignments are shown for the unbound DARC ¹H-¹⁵N-TROSY spectra. Peaks still visible in the presence of DBP-RII (red) are at DARC 1–60's N- and C- termini. Residues that disappear in the presence of DBP-RII are in the center of DARC and delineate the binding region.</p

Crystal Structure of the DBP-RII∶DARC heterotrimer and heterotetramer.

<p>Overview of (<b>A</b>) DBP-RII∶DARC heterotrimer and (<b>B</b>) the DBP-RII∶DARC heterotetramer. Rotated views, (<b>C</b>) and (<b>D</b>), show DARC helices are oriented in parallel in the heterotetramer. DBP-RII monomers are in yellow and green. DARC monomers are in purple and blue.</p

Data collection and refinement statistics.

<p>Values in parentheses are for highest-resolution shell.</p><p>Data were collected on a single crystal for each dataset.</p

A model for attachment during invasion.

<p>An initial binding event is followed by receptor-induced dimerization, as in the DBP-RII∶DARC heterotrimer. This brings a second DBP-RII molecule in close proximity to a second DARC ectodomain in the DARC homodimer. A second binding event creates the DBP-RII∶DARC heterotetramer. DBP-RII molecules are in green and yellow and DARC19–30 molecules are in purple and blue. The DARC homodimer is represented by a homology model. A schematic for the stepwise assembly is shown at the bottom. Closed circle – bound DBP-RII, open circle – unbound DBP-RII.</p