Beáta G. Vértessy

Structural Biology and Regulation of Protein Import into the Nucleus

Proteins are translated in the cytoplasm, but many need to access the nucleus to perform their functions. Understanding how these nuclear proteins are transported through the nuclear envelope and how the import processes are regulated is therefore an important aspect of understanding cell function. Structural biology has played a key role in understanding the molecular events during the transport processes and their regulation, including the recognition of nuclear targeting signals by the corresponding receptors. Here, we review the structural basis of the principal nuclear import pathways and the molecular basis of their regulation. The pathways involve transport factors that are members of the β-karyopherin family, which can bind cargo directly (e.g., importin-β, transportin-1, transportin-3, importin-13) or through adaptor proteins (e.g., importin-α, snurportin-1, symportin-1), as well as unrelated transport factors such as Hikeshi, involved in the transport of heat-shock proteins, and NTF2, involved in the transport of RanGDP. Solenoid proteins feature prominently in these pathways. Nuclear transport factors recognize nuclear targeting signals on the cargo proteins, including the classical nuclear localization signals, recognized by the adaptor importin-α, and the PY nuclear localization signals, recognized by transportin-1. Post-translational modifications, particularly phosphorylation, constitute key regulatory mechanisms operating in these pathways.

Alternative binding of two sequential glycolytic enzymes to microtubules. Molecular studies in the phosphofructokinase/aldolase/microtubule system

Simultaneous binding of two sequential glycolytic enzymes, phosphofructokinase and aldolase, to a microtubular network was investigated. The binding of the phosphofructokinase to microtubules and its bundling activity has been previously characterized (Lehotzky, A., Telegdi, M., Liliom, K., and Ovádi, J. (1993) J. Biol. Chem. 268, 10888–10894). Aldolase binding to microtubules at near physiological ionic strength is weak (K d = 20 μm) as compared with that of the kinase (K d = 1 μm). The interactions of both enzymes with microtubules are modulated by their common intermediate, fructose-1,6-bisphosphate. Pelleting and electron microscopic measurements have revealed that the aldolase binding interferes with that of phosphofructokinase, although they have distinct binding domains on microtubules. The underlying molecular mechanism responsible for this finding is that in the solution phase aldolase and phosphofructokinase form a bienzyme complex that does not bind to the microtubule. The bienzyme complex formation does not influence the catalytic activity of aldolase, however, it inhibits the dissociation-induced inactivation of the kinase by stabilizing a catalytically active molecular form. The present data suggest the first experimental evidence that two sequential glycolytic enzymes do not associate simultaneously to microtubules, but their complexation in solution provides kinetic advantage for glycolysis.

Flexible glycine rich motif of Escherichia coli deoxyuridine triphosphate nucleotidohydrolase is important for functional but not for structural integrity of the enzyme

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a ubiquitous enzyme of DNA metabolism, has been implicated as a novel target of anticancer and antiviral drug design. This task is most efficiently accomplished by X‐ray crystallography of the relevant protein–inhibitor complexes. However, the topic of the present investigation, a glycine‐rich strictly conserved structural motif of dUTPases, could not be located in the crystal structure of the Escherichia coli enzyme, probably due to its increased flexibility. The present work shows that removal of a C‐terminal 11‐residue fragment, including this motif, by limited trypsinolysis strongly impairs catalytic activity. Kinetic analysis of the intact and digested variants showed that kcat decreases 40‐fold, while KM increases less than twofold upon digestion. The tryptic site was identified by mass spectrometry, amino acid analysis and N‐terminal sequencing. The shortened enzyme variant retains the secondary, tertiary, and quaternary (trimeric) structure of the intact species as suggested by UV absorption, fluorescence and circular dichroism spectroscopy, and analytical gel filtration. Moreover, binding affinity of the shortened variant toward the substrate analogue MgdUDP is identical to the one displayed by the intact enzyme. I conclude that the glycine‐rich motif is functionally relevant for E. coli dUTPase. It may play a role in enzymatic catalysis by contributing to the formation of the catalytically potent enzyme–substrate complex. Proteins 28:568–579, 1997.

Active site of mycobacterial dUTPase: structural characteristics and a built-in sensor.

dUTPases are essential to eliminate dUTP for DNA integrity and provide dUMP for thymidylate biosynthesis. Mycobacterium tuberculosis apparently lacks any other thymidylate biosynthesis pathway, therefore dUTPase is a promising antituberculotic drug target. Crystal structure of the mycobacterial enzyme in complex with the isosteric substrate analog, alpha,beta-imido-dUTP and Mg(2+) at 1.5A resolution was determined that visualizes the full-length C-terminus, previously not localized. Interactions of a conserved motif important in catalysis, the Mycobacterium-specific five-residue-loop insert and C-terminal tetrapeptide could now be described in detail. Stacking of C-terminal histidine upon the uracil moiety prompted replacement with tryptophan. The resulting sensitive fluorescent sensor enables fast screening for binding of potential inhibitors to the active site. K(d) for alpha,beta-imido-dUTP binding to mycobacterial dUTPase is determined to be 10-fold less than for human dUTPase, which is to be considered in drug optimization. A robust continuous activity assay for kinetic screening is proposed.

Kinetic Mechanism of Human dUTPase, an Essential Nucleotide Pyrophosphatase Enzyme

Human dUTPase is essential in controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. The nuclear isoform of the enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies. The recently determined three-dimensional structure of this protein in complex with an isosteric substrate analogue allowed in-depth structural characterization of the active site. However, fundamental steps of the dUTPase enzymatic cycle have not yet been revealed. This knowledge is indispensable for a functional understanding of the molecular mechanism and can also contribute to the design of potential antagonists. Here we present detailed pre-steady-state and steady-state kinetic investigations using a single tryptophan fluorophore engineered into the active site of human dUTPase. This sensor allowed distinction of the apoenzyme, enzyme-substrate, and enzyme-product complexes. We show that the dUTP hydrolysis cycle consists of at least four distinct enzymatic steps: (i) fast substrate binding, (ii) isomerization of the enzyme-substrate complex into the catalytically competent conformation, (iii) a hydrolysis (chemical) step, and (iv) rapid, nonordered release of the products. Independent quenched-flow experiments indicate that the chemical step is the rate-limiting step of the enzymatic cycle. To follow the reaction in the quenched-flow, we devised a novel method to synthesize γ-32P-labeled dUTP. We also determined by indicator-based rapid kinetic assays that proton release is concomitant with the rate-limiting hydrolysis step. Our results led to a quantitative kinetic model of the human dUTPase catalytic cycle and to direct assessment of relative flexibilities of the C-terminal arm, critical for enzyme activity, in the enzyme-ligand complexes along the reaction pathway.

Active site closure facilitates juxtaposition of reactant atoms for initiation of catalysis by human dUTPase

Human dUTPase, essential for DNA integrity, is an important survival factor for cancer cells. We determined the crystal structure of the enzyme:α,β‐imino‐dUTP:Mg complex and performed equilibrium binding experiments in solution. Ordering of the C‐terminus upon the active site induces close juxtaposition of the incoming nucleophile attacker water oxygen and the α‐phosphorus of the substrate, decreasing their distance below the van der Waals limit. Complex interactions of the C‐terminus with both substrate and product were observed via a specifically designed tryptophan sensor, suitable for further detailed kinetic and ligand binding studies. Results explain the key functional role of the C‐terminus.

Flexible segments modulate co-folding of dUTPase and nucleocapsid proteins

The homotrimeric fusion protein nucleocapsid (NC)-dUTPase combines domains that participate in RNA/DNA folding, reverse transcription, and DNA repair in Mason-Pfizer monkey betaretrovirus infected cells. The structural organization of the fusion protein remained obscured by the N- and C-terminal flexible segments of dUTPase and the linker region connecting the two domains that are invisible in electron density maps. Small-angle X-ray scattering reveals that upon oligonucleotide binding the NC domains adopt the trimeric symmetry of dUTPase. High-resolution X-ray structures together with molecular modeling indicate that fusion with NC domains dramatically alters the conformation of the flexible C-terminus by perturbing the orientation of a critical β-strand. Consequently, the C-terminal segment is capable of double backing upon the active site of its own monomer and stabilized by non-covalent interactions formed with the N-terminal segment. This co-folding of the dUTPase terminal segments, not observable in other homologous enzymes, is due to the presence of the fused NC domain. Structural and genomic advantages of fusing the NC domain to a shortened dUTPase in betaretroviruses and the possible physiological consequences are envisaged.

The complete triphosphate moiety of non-hydrolyzable substrate analogues is required for a conformational shift of the flexible C-terminus in E. coli dUTP pyrophosphatase

The molecular mechanism of substrate analogue interaction with Escherichia coli dUTPase was investigated, using the non‐hydrolyzable 2′‐deoxyuridine 5′‐(α,β‐imido)triphosphate (α,β‐imido‐dUTP). Binding of this analogue induces a difference in the far UV circular dichroism (CD) spectrum arguing for a significant change in protein conformation. The spectral shift is strictly Mg2+‐dependent, does not appear with dUDP instead of α,β‐imido‐dUTP and is not elicited if the flexible C‐terminal arm is deleted from the protein by limited tryptic digestion. Involvement of the C‐terminal arm in α,β‐imido‐dUTP binding is consistent with the finding that this analogue protects against tryptic hydrolysis at Arg‐141. Near UV CD of ligand‐enzyme complexes reveals a characteristic difference in the microenvironments of enzyme‐bound dUDP and α,β‐imido‐dUTP, a difference not observable in C‐terminally truncated dUTPase. The results suggest that (i) closing of the active site during the catalytic cycle, through the movement of the C‐terminal arm, requires the presence of the complete triphosphate moiety of the substrate in complex with Mg2+, and (ii) after catalytic cleavage the active site pops open to facilitate product release.

Aromatic stacking between nucleobase and enzyme promotes phosphate ester hydrolysis in dUTPase

Aromatic interactions are well-known players in molecular recognition but their catalytic role in biological systems is less documented. Here, we report that a conserved aromatic stacking interaction between dUTPase and its nucleotide substrate largely contributes to the stabilization of the associative type transition state of the nucleotide hydrolysis reaction. The effect of the aromatic stacking on catalysis is peculiar in that uracil, the aromatic moiety influenced by the aromatic interaction is relatively distant from the site of hydrolysis at the alpha-phosphate group. Using crystallographic, kinetics, optical spectroscopy and thermodynamics calculation approaches we delineate a possible mechanism by which rate acceleration is achieved through the remote π–π interaction. The abundance of similarly positioned aromatic interactions in various nucleotide hydrolyzing enzymes (e.g. most families of ATPases) raises the possibility of the reported phenomenon being a general component of the enzymatic catalysis of phosphate ester hydrolysis.

Malaria pigment crystals as magnetic micro-rotors: key for high-sensitivity diagnosis

The need to develop new methods for the high-sensitivity diagnosis of malaria has initiated a global activity in medical and interdisciplinary sciences. Most of the diverse variety of emerging techniques are based on research-grade instruments, sophisticated reagent-based assays or rely on expertise. Here, we suggest an alternative optical methodology with an easy-to-use and cost-effective instrumentation based on unique properties of malaria pigment reported previously and determined quantitatively in the present study. Malaria pigment, also called hemozoin, is an insoluble microcrystalline form of heme. These crystallites show remarkable magnetic and optical anisotropy distinctly from any other components of blood. As a consequence, they can simultaneously act as magnetically driven micro-rotors and spinning polarizers in suspensions. These properties can gain importance not only in malaria diagnosis and therapies, where hemozoin is considered as drug target or immune modulator, but also in the magnetic manipulation of cells and tissues on the microscopic scale.