Structure-Based Prototyping of Allosteric Inhibitors of Human Uridine/Cytidine Kinase 2 (UCK2)

Pyrimidine nucleotide biosynthesis in humans is a promising chemotherapeutic target for infectious diseases caused by RNA viruses. Because mammalian cells derive pyrimidine ribonucleotides through a combination of de novo biosynthesis and salvage, combined inhibition of dihydroorotate dehydrogenase (DHODH; the first committed step in de novo pyrimidine nucleotide biosynthesis) and uridine/cytidine kinase 2 (UCK2; the first step in salvage of exogenous nucleosides) strongly attenuates viral replication in infected cells. However, while several pharmacologically promising inhibitors of human DHODH are known, to date there are no reports of medicinally viable leads against UCK2. Here, we use structure-based drug prototyping to identify two classes of promising leads that noncompetitively inhibit UCK2 activity. In the process, we have identified a hitherto unknown allosteric site at the intersubunit interface of this homotetrameric enzyme. By reducing the kcat of human UCK2 without altering its KM, these new inhibitors have the potential to enable systematic dialing of the fractional inhibition of pyrimidine salvage to achieve the desired antiviral effect with minimal host toxicity

Point mutations in DivL's canonical HK-RR binding interface disrupt DivL-DivK∼P binding <i>in vitro</i>.

<p>(A) A computational model of the DivL-DivK∼P interface based on the HK853 complex with its cognate RD RR468 (PDB code 3dge). In this close-up view of the DivL-DivK∼P interaction DivK (PDB code 1mav) is shown as a white surface in the background with residues contacting DivL highlighted in yellow. The two forms of DivL are illustrated as violet (DivL-A) and cyan (DivL-B) Cα traces. Residues selected for site-directed mutagenesis are highlighted in green on DivL-A. (B) Fluorescence polarization binding assay examining the impact of point mutations (Y550H, R553A, T557N, Y562A, and H579E) on DivL-DivK and DivL-DivK∼P binding interaction. DivL variants R553A, T557N, Y562A, and H579E disrupt the binding to DivK, while DivL variant Y550H has no impact on DivK∼P binding. Numerical data used to generate manuscript graphs or histograms can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001979#pbio.1001979.s010" target="_blank">Table S1</a>.</p

Cell Fate Regulation Governed by a Repurposed Bacterial Histidine Kinase

<div><p>One of the simplest organisms to divide asymmetrically is the bacterium <i>Caulobacter crescentus</i>. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions <i>in vivo</i> without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK∼P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.</p></div

DivL sensor domains are critical for phosphospecific DivK recognition.

<p>(A) DivK (black) versus DivK∼P (red) binding curves for the DivL DHp-CA construct (no PAS domains) versus the DivL PAS 3X-DHp-CA construct (three PAS domains). (B) Relative amounts of DivK∼P versus DivK binding to DivL constructs containing 3, 2, 1, or 0 PAS domains. The minimal DivL construct that maintains phosphospecific DivK recognition contains two PAS domains. No DivL-DivK or DivL-DivK∼P binding was detected for the 1-PAS domain DivL construct. (C) Position of the allosteric point mutation A601L at the input-output helix interface. (D) Fluorescence polarization binding assay examining the impact of DivL point mutations in the DivL DHp-CA construct (black, left) versus the DivL PAS 3X-DHp-CA construct (red, right) upon both DivK and DivK∼P binding. The DivL(A601L) variant binds DivK∼P, but only when the PAS domains are deleted. Numerical data used to generate manuscript graphs or histograms can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001979#pbio.1001979.s010" target="_blank">Table S1</a>.</p

Model for a repurposed pseudokinase that functions as a sensor for a phosphorylated RR.

<p>In conventional two-component signaling systems, an HK receives a signal in the sensor region and ultimately promotes phosphorylation of a response regulator. In contrast, DivL repurposes its RR docking surface and canonical output domain as a sensory module for DivK∼P. Upon binding DivK∼P, DivL causes the repression of the CtrA differentiation pathway, potentially through conformational remodeling of the DivL PAS sensor domains.</p

The DivL structure reveals an asymmetric dimer with conformational difference of monomers described as substructure rigid-body movements.

<p>(A) The DivL dimer. The monomeric subunits DivL-A and DivL-B are colored in violet and cyan, respectively. Tyr550 (yellow) and Ala601 (blue) are shown as sticks. (B) A monomer of DivL (here, DivL-A) consists of three rigid-body substructures (the “output helix+CA” module, input helix, and RR docking module, shaded in gray). (C) A stereoview of two DivL monomers superimposed about the RR docking module. Interatomic distances (in Å) are represented with dashed lines. (D) A schematic diagram showing the structural differences in two monomers (DivL-A and DivL-B) with substructures colored red (input helix), orange (RR docking module), and yellow (output helix+CA domain).</p

A compartment sensing circuit regulates asymmetric cell division in <i>Caulobacter crescentus</i>.

<p>(A) A cartoon of <i>C. crescentus</i> asymmetric cell division and the spatiotemporal localization of the essential regulatory components DivJ (blue), DivK (red), PleC (orange), CckA (brown), DivL (green), and CtrA (yellow). PleC serves as a DivK phosphatase localized at the new cell pole, while DivJ serves as a DivK kinase localized at the stalked cell pole. DivJ and PleC are physically separated upon compartmentalization, generating DivK∼P (full red circles) in the stalk compartment and DivK (open red circles) in the swarmer compartment. This compartment signal (DivK) selectively represses CtrA upon interaction with DivL thereby guiding distinct developmental programs for swarmer and stalked cells. (B) To promote cell differentiation, <i>Caulobacter</i> employs three distinct signaling modules: a compartment sensing module consisting of DivJ (HK), DivK (RR), and PleC (phosphatase), a differentiation module consisting of CckA (hybrid HK), ChpT (phosphotransferase), and CtrA (RR), and a cell-fate-switch module comprising the DivL pseudokinase that connects the compartment-sensing and differentiation modules. DivL activates the differentiation module when unbound (via CckA), but represses this module when bound to DivK∼P. (C) The domain organization of DivL in select α-proteobacteria that contain a putative DivK ortholog versus DivL domain organization in α-proteobacteria that do not contain a putative DivK ortholog <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001979#pbio.1001979-Brilli1" target="_blank">[19]</a>. Based on comparative analysis, DivL(523–769) containing only the DHp-CA domains was selected as a minimal DivK interacting unit for crystallization trials.</p

Functional interrogation of the DivL-DivK∼P interaction.

<p>(A) DivL-mediated stabilization of DivK∼P versus PleC-mediated dephosphorylation of DivK∼P is shown through a dephosphorylation time course (lane assignments: 1, 10, 20, 60, 120, 180, 240 minutes) with purified DivK∼P (1 µM) incubated alone, or together with 2.5 µM PleC or 2.5 µM DivL. Samples were transferred into SDS sample buffer and subjected to electrophoresis followed by phosphorimaging. (B) Quantification of DivK∼P decay from (A) in reactions containing DivK∼P alone (black) or DivK∼P with PleC (orange), or DivL (green), or DivL Y550H (red). (C) DivK stimulates DivJ ATP consumption, but has no impact on DivL ATP consumption. ATP consumption rates for DivJ, DivJ-DivK, DivL alone, and DivL-DivK are shown. (D) DivK∼P stimulates DivL Y550H autophosphorylation but has no impact on the phosphorylation state of wild-type DivL. Purified DivK∼P and [γ-³²P]ATP were incubated with 5 µM PleC, with 5 µM DivL, or with 5 µM DivL Y550H for 0, 0.5, 1, 2, 5, and 20 minutes. (E) Cartoon illustrating the functional differences between the PleC phosphatase and the DivL phosphospecific DivK∼P sensor. Samples were transferred into SDS sample buffer and subjected to electrophoresis followed by phosphorimaging. Numerical data used to generate manuscript graphs or histograms can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001979#pbio.1001979.s010" target="_blank">Table S1</a>.</p

DivK binds specifically to PleC, DivJ, and DivL.

<p>Fluorescence polarization binding assay of BODIPY dye labeled DivK under unphosphorylated (gray) versus phosphorylated (black) conditions mixed with the following HKs at 10 µM PleC, DivJ, DivL, CckA, and ChpT. PleC and DivL binds to phosphorylated DivK specifically, while DivJ binds to both phosphorylated and unphosphorylated DivK. Numerical data used to generate manuscript graphs or histograms can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001979#pbio.1001979.s010" target="_blank">Table S1</a>.</p

Impact of DivL point mutations on cell fitness.

<p>(A) Genetic model describing the functional consequences of DivL-DivK∼P binding on <i>Caulobacter</i> growth and development. Critically over-active CtrA impacts cell division, motility, and DivL subcellular localization. (B) Morphology assay of <i>Caulobacter</i> strains grown in PYE indicates that Y550H, R553A and A601L display cell filamentation and cell division defects, while the Y562A variant is similar in morphology to a wild-type strain. (C) An efficiency of plating assay indicates a reduction in growth rates of Y550H, R553A, and A601L, while Y562A displayed a mild reduction in growth rate relative to wild-type.</p