The Structure of Thymidylate Kinase from <i>Candida albicans</i> Reveals a Unique Structural Element

The structure of thymidylate kinase from <i>Candida albicans</i>, determined by X-ray crystallography, is reported to a resolution of 2.45 Å with a final <i>R</i>_free of 0.223. Thymidylate kinase from <i>C. albicans</i> possesses a unique 15-residue loop that is not seen in thymidylate kinases from other genera. The structure reported here reveals that the conformation of this loop is constrained by both intra- and intersubunit hydrogen bonding, and a number of key residues in this loop are conserved among different <i>Candida</i> species that are medically important. The substrate specificity of the enzyme was determined using a novel nuclear magnetic resonance-based assay as well as a traditional coupled assay. The enzyme is active against 3′-azido-3′-deoxythymidine monophosphate and moderately active with dGMP. The distinct functional and structural differences between the <i>C. albicans</i> enzyme and the human enzyme suggest that thymidylate kinase is an appropriate target for the development of new antifungal agents

Specific Labeling of Threonine Methyl Groups for NMR Studies of Protein–Nucleic Acid Complexes

Specific ¹³C labeling of Thr methyl groups has been accomplished via the growth of a standard laboratory strain of <i>Escherichia coli</i> on [2-¹³C]­glycerol in the presence of deuterated isoketovalerate, Ile, and Ala. Diversion of the label from the Thr biosynthetic pathway is suppressed by including Lys, Met, and Ile in the growth medium. This method complements the repertoire of methyl labeling schemes for NMR structural and dynamic studies of proteins and is particularly useful for the study of nucleic acid binding proteins because of the high propensity of Thr residues at protein–DNA and −RNA interfaces

Dual Lifetimes for Complexes between Glutathione‑<i>S</i>‑transferase (hGSTA1-1) and Product-like Ligands Detected by Single-Molecule Fluorescence Imaging

Single-molecule fluorescence techniques were used to characterize the binding of products and inhibitors to human glutathione <i>S</i>-transferase A1-1 (hGSTA1-1). The identification of at least two different bound states for the wild-type enzyme suggests that there are at least two conformations of the protein, consistent with the model that ligand binding promotes closure of the carboxy-terminal helix over the active site. Ligand induced changes in ensemble fluorescence energy transfer support this proposed structural change. The more predominant state in the ensemble of single molecules shows a significantly faster off-rate, suggesting that the carboxy-terminal helix is delocalized in this state, permitting faster exit of the bound ligand. A point mutation (I219A), which is known to interfere with the association of the carboxy-terminal helix with the enzyme, shows increased rates of interconversion between the open and closed state. Kinematic traces of fluorescence from single molecules show that a single molecule readily samples a number of different conformations, each with a characteristic off-rate

Interaction of α‑Thymidine Inhibitors with Thymidylate Kinase from <i>Plasmodium falciparum</i>

<i>Plasmodium falciparum</i> thymidylate kinase (PfTMK) is a critical enzyme in the <i>de novo</i> biosynthesis pathway of pyrimidine nucleotides. <i>N</i>-(5′-Deoxy-α-thymidin-5′-yl)-<i>N</i>′-[4-(2-chlorobenzyloxy)­phenyl]­urea was developed as an inhibitor of PfTMK and has been reported as an effective inhibitor of <i>P. falciparum</i> growth with an EC₅₀ of 28 nM [Cui, H., et al. (2012) <i>J. Med. Chem. 55</i>, 10948–10957]. Using this compound as a scaffold, a number of derivatives were developed and, along with the original compound, were characterized in terms of their enzyme inhibition (<i>K</i>_i) and binding affinity (<i>K</i>_D). Furthermore, the binding site of the synthesized compounds was investigated by a combination of mutagenesis and docking simulations. Although the reported compound is indicated to be highly effective in its inhibition of parasite growth, we observed significantly lower binding affinity and weaker inhibition of PfTMK than expected from the reported EC₅₀. This suggests that significant structural optimization will be required for the use of this scaffold as an effective PfTMK inhibitor and that the inhibition of parasite growth is due to an off-target effect

Metal Ion Binding at the Catalytic Site Induces Widely Distributed Changes in a Sequence Specific Protein–DNA Complex

Metal ion cofactors can alter the energetics and specificity of sequence specific protein–DNA interactions, but it is unknown if the underlying effects on structure and dynamics are local or dispersed throughout the protein–DNA complex. This work uses EcoRV endonuclease as a model, and catalytically inactive lanthanide ions, which replace the Mg²⁺ cofactor. Nuclear magnetic resonance (NMR) titrations indicate that four Lu³⁺ or two La³⁺ cations bind, and two new crystal structures confirm that Lu³⁺ binding is confined to the active sites. NMR spectra show that the metal-free EcoRV complex with cognate (GATATC) DNA is structurally distinct from the nonspecific complex, and that metal ion binding sites are not assembled in the nonspecific complex. NMR chemical shift perturbations were determined for ¹H–¹⁵N amide resonances, for ¹H–¹³C Ile-δ-CH₃ resonances, and for stereospecifically assigned Leu-δ-CH₃ and Val-γ-CH₃ resonances. Many chemical shifts throughout the cognate complex are unperturbed, so metal binding does not induce major conformational changes. However, some large perturbations of amide and side chain methyl resonances occur as far as 34 Å from the metal ions. Concerted changes in specific residues imply that local effects of metal binding are propagated via a β-sheet and an α-helix. Both amide and methyl resonance perturbations indicate changes in the interface between subunits of the EcoRV homodimer. Bound metal ions also affect amide hydrogen exchange rates for distant residues, including a distant subdomain that contacts DNA phosphates and promotes DNA bending, showing that metal ions in the active sites, which relieve electrostatic repulsion between protein and DNA, cause changes in slow dynamics throughout the complex

Metal Ion Binding at the Catalytic Site Induces Widely Distributed Changes in a Sequence Specific Protein–DNA Complex

Metal ion cofactors can alter the energetics and specificity of sequence specific protein–DNA interactions, but it is unknown if the underlying effects on structure and dynamics are local or dispersed throughout the protein–DNA complex. This work uses EcoRV endonuclease as a model, and catalytically inactive lanthanide ions, which replace the Mg²⁺ cofactor. Nuclear magnetic resonance (NMR) titrations indicate that four Lu³⁺ or two La³⁺ cations bind, and two new crystal structures confirm that Lu³⁺ binding is confined to the active sites. NMR spectra show that the metal-free EcoRV complex with cognate (GATATC) DNA is structurally distinct from the nonspecific complex, and that metal ion binding sites are not assembled in the nonspecific complex. NMR chemical shift perturbations were determined for ¹H–¹⁵N amide resonances, for ¹H–¹³C Ile-δ-CH₃ resonances, and for stereospecifically assigned Leu-δ-CH₃ and Val-γ-CH₃ resonances. Many chemical shifts throughout the cognate complex are unperturbed, so metal binding does not induce major conformational changes. However, some large perturbations of amide and side chain methyl resonances occur as far as 34 Å from the metal ions. Concerted changes in specific residues imply that local effects of metal binding are propagated via a β-sheet and an α-helix. Both amide and methyl resonance perturbations indicate changes in the interface between subunits of the EcoRV homodimer. Bound metal ions also affect amide hydrogen exchange rates for distant residues, including a distant subdomain that contacts DNA phosphates and promotes DNA bending, showing that metal ions in the active sites, which relieve electrostatic repulsion between protein and DNA, cause changes in slow dynamics throughout the complex

A Variable Light Domain Fluorogen Activating Protein Homodimerizes To Activate Dimethylindole Red

Novel fluorescent tools such as green fluorescent protein analogues and fluorogen activating proteins (FAPs) are useful in biological imaging for tracking protein dynamics in real time with a low fluorescence background. FAPs are single-chain variable fragments (scFvs) selected from a yeast surface display library that produce fluorescence upon binding a specific dye or fluorogen that is normally not fluorescent when present in solution. FAPs generally consist of human immunoglobulin variable heavy (V_H) and variable light (V_L) domains covalently attached via a glycine- and serine-rich linker. Previously, we determined that the yeast surface clone, V_H-V_L M8, could bind and activate the fluorogen dimethylindole red (DIR) but that the fluorogen activation properties were localized to the M8V_L domain. We report here that both nuclear magnetic resonance and X-ray diffraction methods indicate the M8V_L forms noncovalent, antiparallel homodimers that are the fluorogen activating species. The M8V_L homodimers activate DIR by restriction of internal rotation of the bound dye. These structural results, together with directed evolution experiments with both V_H-V_L M8 and M8V_L, led us to rationally design tandem, covalent homodimers of M8V_L domains joined by a flexible linker that have a high affinity for DIR and good quantum yields

Design of Bivalent Nucleic Acid Ligands for Recognition of RNA-Repeated Expansion Associated with Huntington’s Disease

We report the development of a new class of nucleic acid ligands that is comprised of Janus bases and the MPγPNA backbone and is capable of binding rCAG repeats in a sequence-specific and selective manner via, inference, bivalent H-bonding interactions. Individually, the interactions between ligands and RNA are weak and transient. However, upon the installation of a C-terminal thioester and an N-terminal cystine and the reduction of disulfide bond, they undergo template-directed native chemical ligation to form concatenated oligomeric products that bind tightly to the RNA template. In the absence of an RNA target, they self-deactivate by undergoing an intramolecular reaction to form cyclic products, rendering them inactive for further binding. The work has implications for the design of ultrashort nucleic acid ligands for targeting rCAG-repeat expansion associated with Huntington’s disease and a number of other related neuromuscular and neurodegenerative disorders