High-resolution X-ray Crystallographic Data Research Articles

Reaction of O2 with a diiron protein generates a mixed-valent Fe2+/Fe3+ center and peroxide

The gene encoding the cyanobacterial ferritin SynFtn is up-regulated in response to copper stress. Here, we show that, while SynFtn does not interact directly with copper, it is highly unusual in several ways. First, its catalytic diiron ferroxidase center is unlike those of all other characterized prokaryotic ferritins and instead resembles an animal H-chain ferritin center. Second, as demonstrated by kinetic, spectroscopic, and high-resolution X-ray crystallographic data, reaction of O2 with the di-Fe2+ center results in a direct, one-electron oxidation to a mixed-valent Fe2+/Fe3+ form. Iron-O2 chemistry of this type is currently unknown among the growing family of proteins that bind a diiron site within a four α-helical bundle in general and ferritins in particular. The mixed-valent form, which slowly oxidized to the more usual di-Fe3+ form, is an intermediate that is continually generated during mineralization. Peroxide, rather than superoxide, is shown to be the product of O2 reduction, implying that ferroxidase centers function in pairs via long-range electron transfer through the protein resulting in reduction of O2 bound at only one of the centers. We show that electron transfer is mediated by the transient formation of a radical on Tyr40, which lies ∼4 Å from the diiron center. As well as demonstrating an expansion of the iron-O2 chemistry known to occur in nature, these data are also highly relevant to the question of whether all ferritins mineralize iron via a common mechanism, providing unequivocal proof that they do not.

Mycobacterium Cytidylate Kinase Appears to Be an Undruggable Target

New and improved drugs against tuberculosis are urgently needed as multi-drug-resistant forms of the disease become more prevalent. Mycobacterium tuberculosis cytidylate kinase is an attractive target for screening due to its essentiality and different substrate specificity to the human orthologue. However, we selected the Mycobacterium smegmatis cytidylate kinase for screening because of the availability of high-resolution X-ray crystallographic data defining its structure and the high likelihood of active site structural similarity to the M. tuberculosis orthologue. We report the development and implementation of a high-throughput luciferase-based activity assay and screening of 19,920 compounds derived from small-molecule libraries and an in silico screen predicting likely inhibitors of the cytidylate kinase enzyme. Hit validation included a counterscreen for luciferase inhibitors that would result in false positives in the initial screen. Results of this counterscreen ruled out all of the putative cytidylate kinase inhibitors identified in the initial screening, leaving no compounds as candidates for drug development. Although a negative result, this study indicates that this important drug target may in fact be undruggable and serve as a warning for future investigations.

Molecular Dynamics of Protein Kinase-Inhibitor Complexes: A Valid Structural Information

Protein kinases (PKs) are key components of protein phosphorylation based signaling networks in eukaryotic cells. They have been identified as being implicated in many diseases. High-resolution X-ray crystallographic data exist for many PKs and, in many cases, these structures are co-complexed with inhibitors. Although this valuable information confirms the precise structure of PKs and their complexes, it ignores the dynamic movements of the structures which are relevant to explain the affinities and selectivity of the ligands, to characterize the thermodynamics of the solvated complexes, and to derive predictive models. Atomistic molecular dynamics (MD) simulations present a convenient way to study PK-inhibitor complexes and have been increasingly used in recent years in structure-based drug design. MD is a very useful computational method and a great counterpart for experimentalists, which helps them to derive important additional molecular information. That enables them to follow and understand structure and dynamics of protein-ligand systems with extreme molecular detail on scales where motion of individual atoms can be tracked. MD can be used to sample dynamic molecular processes, and can be complemented with more advanced computational methods (e.g., free energy calculations, structure-activity relationship analysis). This review focuses on the most commonly applications to study PK-inhibitor complexes using MD simulations. Our aim is that researchers working in the design of PK inhibitors be aware of the benefits of this powerful tool in the design of potent and selective PK inhibitors.

The Amino Acid-Polyamine-Organocation Superfamily

The amino acid-polyamine-organocation (APC) superfamily has been shown to include five recognized families, four of which are specific for amino acids and their derivatives. Recent high-resolution X-ray crystallographic data have shown that four additional transporter families (BCCT, TC No. 2.A.15; SSS, 2.A.21; NSS, 2.A.22; and NCS1, 2.A.39), transporting a wide range of solutes, exhibit sufficiently similar folds to suggest a common evolutionary origin. We have used established statistical methods, based on sequence similarity, to show that these families are, in fact, members of the APC superfamily. We also identify two additional families (NCS2, 2.A.40; SulP, 2.A.53) as being members of this superfamily. Repeat sequences, each having five transmembrane α-helical segments and arising via ancient intragenic duplications, are demonstrated for all of these families, further strengthening the conclusion of homology. The APC superfamily appears to be the second largest superfamily of secondary carriers, the largest being the major facilitator superfamily (MFS). Although the topology of the members of the APC superfamily differs from that of the MFS, both families appear to have arisen from a common ancestral 2 TMS hairpin structure that underwent intragenic triplication followed by loss of a TMS in the APC family, to give the repeat units that are characteristic of these two superfamilies.

Crystal structure of a high-affinity variant of rat alpha-parvalbumin.

In model peptide systems, Ca2+ affinity is maximized in EF-hand motifs containing four carboxylates positioned on the +x and -x and +z and -z axes; introduction of a fifth carboxylate ligand reduces the affinity. However, in rat beta-parvalbumin, replacement of Ser-55 with aspartate heightens divalent ion affinity [Henzl, M. T., et al. (1996) Biochemistry 35, 5856-5869]. The corresponding alpha-parvalbumin variant (S55D/E59D) likewise exhibits elevated affinity [Henzl, M. T., et al. (2003) Anal. Biochem. 319, 216-233]. To determine whether these mutations produce a variation on the archetypal EF-hand coordination scheme, we have obtained high-resolution X-ray crystallographic data for alpha S55D/E59D. As anticipated, the aspartyl carboxylate replaces the serine hydroxyl at the +z coordination position. Interestingly, the Asp-59 carboxylate abandons the role it plays as an outer sphere ligand in wild-type rat beta, rotating away from the Ca2+ and, instead, forming a hydrogen bond with the amide of Glu-62. Superficially, the coordination sphere in the CD site of alpha S55D/E59D resembles that in the EF site. However, the orientation of the Asp-59 side chain is predicted to stabilize the D-helix, which may contribute to the heightened divalent ion affinity. DSC data indicate that the alpha S55D/E59D variant retains the capacity to bind 1 equiv of Na+. Consistent with this finding, when binding measurements are conducted in K(+)-containing buffer, divalent ion affinity is markedly higher. In 0.15 M KCl and 0.025 M Hepes-KOH (pH 7.4) at 5 degrees C, the macroscopic Ca2+ binding constants are 1.8 x 10(10) and 2.0 x 10(9) M(-1). The corresponding Mg2+ binding constants are 2.7 x 10(6) and 1.2 x 10(5) M(-1).

The RNA polymerase structure-function study (1962-2001)

The study of RNA polymerase initiated by R.B. Khesin has been conducted for about forty years at the laboratory founded by him (since 1989, in collaboration with A. Goldfarb's laboratory). Genetic methods are used in combination with methods of the specific chemical crosslinks of nucleic acids with proteins. The paper assesses the main results of the study in comparison with the recent high-resolution X-ray crystallographic data. A short comparative summary of the RNA polymerase structure is presented for bacteria, archaebacteria, and eukaryotic organelles and nuclei. A brief history of the RNA polymerase study is also given.

Identification of dipeptidyl nitriles as potent and selective inhibitors of cathepsin B through structure-based drug design.

Cathepsin B is a member of the papain superfamily of cysteine proteases and has been implicated in the pathology of numerous diseases, including arthritis and cancer. As part of an effort to identify potent, reversible inhibitors of this protease, we examined a series of dipeptidyl nitriles, starting with the previously reported Cbz-Phe-NH-CH(2)CN (19, IC(50) = 62 microM). High-resolution X-ray crystallographic data and molecular modeling were used to optimize the P(1), P(2), and P(3) substituents of this template. Cathepsin B is unique in its class in that it contains a carboxylate recognition site in the S(2)' pocket of the active site. Inhibitor potency and selectivity were enhanced by tethering a carboxylate functionality from the carbon alpha to the nitrile to interact with this region of the enzyme. This resulted in the identification of compound 10, a 7 nM inhibitor of cathepsin B, with excellent selectivity over other cysteine cathepsins.

Thermodynamics of Metal Ion Binding. 1. Metal Ion Binding by Wild-Type Carbonic Anhydrase

Understanding the energetic consequences of molecular structure in aqueous solution is a prerequisite to the rational design of synthetic motifs with predictable properties. Such properties include ligand binding and the collapse of polymer chains into discrete three-dimensional structures. Despite advances in macromolecular structure determination, correlations of structure with high-resolution thermodynamic data remain limited. Here we compare thermodynamic parameters for the binding of Zn(II), Cu(II), and Co(II) to human carbonic anhydrase II. These calorimetrically determined values are interpreted in terms of high-resolution X-ray crystallographic data. While both zinc and cobalt are bound with a 1:1 stoichiometry, CAII binds two copper ions. Considering only the high-affinity site, there is a diminution in the enthalpy of binding through the series Co(II) --> Zn(II) --> Cu(II) that mirrors the enthalpy of hydration; this observation reinforces the notion that the thermodynamics of solute association with water is at least as important as the thermodynamics of solute-solute interaction and that these effects must be considered when interpreting association in aqueous solution. Additionally, DeltaC(p) data suggest that zinc binding to CAII proceeds with a greater contribution from desolvation than does binding of either copper or cobalt, suggesting Nature optimizes binding by optimizing desolvation.

Structural and thermodynamic aspects of cooperativity in the homodimeric hemoglobin from Scapharca inaequivalvis

The homodimeric cooperative hemoglobin from the mollusk Scapharca inaequivalvis displays an unusual subunit assembly with respect to vertebrate hemoglobins. The intersubunit contact region is formed by the two heme-carrying E and F helices, which bring the two hemes in contact with each other. At variance with tetrameric vertebrate hemoglobins, the ligand binding is not accompanied by a significant quaternary transition. The major ligand-linked changes are tertiary and are limited to the heme pocket and subunit interface. These unique structural features of HbI are not easily reconciled with the classical thermodynamic models used to describe cooperative ligand binding in vertebrate hemoglobins. The lack of distinct quaternary states and the absence of allosteric effectors suggested that cooperativity in HbI is entirely homotropic in origin. Thereafter, high resolution X-ray crystallographic data displayed the preferential binding of water molecules at the intersubunit interface in the unliganded protein with respect to the liganded one. These ordered water molecules were thus proposed to act as heterotropic effectors in HbI. The contribution of specific water binding to the observed cooperativity in HbI is discussed in the framework of the enthalpy–entropy compensation effect emerging from previous accurate equilibrium oxygen binding measurements.

Spectral changes accompanying the interaction between metal ligands and concanavalin A

The lectin, concanavalin A (con A), is known to have at least four distinct binding sites. On site binds a transition metal, which, when occupied, activates a calcium binding site [ 11. When both metal binding sites are filed, a saccharide binding site is activated. In addition, recent evidence suggests that con A binds a variety of non-polar compounds [2]. Nuclear magnetic resonance and high resolution X-ray crystallographic data [3-61 have shown that metal ion, saccharide and hydrophobic group binding sites are spatially distinct. Since aromatic residues are either coordinated to metals or are in close proximity to the metal binding sites [4,5] one might predict that the interaction of metal ligands with con A would create perturbations which could be detected spectrally. Spectral changes could then be used to study the binding and kinetics of the metal-con A interaction. In this report, we show that the metals associated with native con A, Mn2+ and Ca2+, induce ultraviolet difference spectra in the lectin. Advantage is taken of these spectral changes to titrate con A with calcium.