Vladimir Z. Pletnev

Far-red fluorescent tags for protein imaging in living tissues

A vast colour palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility and interactions. However, low brightness has remained a problem in far-red variants, which hampers multicolour labelling and whole-body imaging techniques. In the present paper, we report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high-brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. We also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-IR fluorescence compared with mRaspberry or mCherry, and is 20-fold brighter than mPlum. Together, monomeric mKate2 and pseudo-monomeric tdKatushka2 represent the next generation of extra-bright far-red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals.

Structure of human estrogenic 17β-hydroxysteroid dehydrogenase at 2.20 å resolution

BACKGROUND The principal human estrogen, 17 beta-estradiol, is a potent stimulator of certain endocrine-dependent forms of breast cancer. Because human estrogenic 17 beta-hydroxysteroid dehydrogenase (type I 17 beta-HSD) catalyzes the last step in the biosynthesis of 17 beta-estradiol from the less potent estrogen, estrone, it is an attractive target for the design of inhibitors of estrogen production and tumor growth. This human enzyme shares less than 15% sequence identity with a bacterial 3 alpha,20 beta-HSD, for which the three-dimensional structure is known. The amino acid sequence of 17 beta-HSD also differs from that of bacterial 3 alpha,20 beta-HSD by two insertions (of 11 and 14 residues) and 52 additional residues at the C terminus. RESULTS The 2.20 A resolution structure of type I 17 beta-HSD, the first mammalian steroidogenic enzyme studied by X-ray crystallographic techniques, reveals a fold characteristic of the short-chain dehydrogenases. The active site contains a Tyr-X-X-X-Lys sequence (where X is any amino acid) and a serine residue, features that are conserved in short-chain steroid dehydrogenases. The structure also contains three alpha-helices and a helix-turn-helix motif, not observed in short-chain dehydrogenase structures reported previously. No cofactor density could be located. CONCLUSIONS The helices present in 17 beta-HSD that were not in the two previous short-chain dehydrogenase structures are located at one end of the substrate-binding cleft away from the catalytic triad. These helices restrict access to the active site and appear to influence substrate specificity. Modeling the position of estradiol in the active site suggests that a histidine side chain may play a critical role in substrate recognition. One or more of these helices may also be involved in the reported association of the enzyme with membranes. A model for steroid and cofactor binding as well as for the estrone to estradiol transition state is proposed. The structure of the active site provides a rational basis for designing more specific inhibitors of this breast cancer associated enzyme.

Structure of uncomplexed and linoleate-bound Candida cylindracea cholesterol esterase.

BACKGROUND Candida cylindracea cholesterol esterase (CE) reversibly hydrolyzes cholesteryl linoleate and oleate. CE belongs to the same alpha/beta hydrolase superfamily as triacylglycerol acyl hydrolases and cholinesterases. Other members of the family that have been studied by X-ray crystallography include Torpedo californica acetylcholinesterase, Geotrichum candidum lipase and Candida rugosa lipase. CE is homologous to C. rugosa lipase 1, a triacylglycerol acyl hydrolase, with which it shares 89% sequence identity. The present study explores the details of dimer formation of CE and the basis for its substrate specificity. RESULTS The structures of uncomplexed and linoleate-bound CE determined at 1.9 A and 2.0 A resolution, respectively, reveal a dimeric association of monomers in which two active-site gorges face each other, shielding hydrophobic surfaces from the aqueous environment. The fatty-acid chain is buried in a deep hydrophobic pocket near the active site. The positioning of the cholesteryl moiety of the substrate is equivocal, but could be modeled in the hydrophobic core of the dimer interface. CONCLUSIONS The monomer structure is the same in both the complexed and uncomplexed crystal forms. The dimers differ in the relative positions of the two monomers at the dimer interface. Of the 55 residues that are different in CE from those in C. rugosa lipase 1, 23 are located in the active site and at the dimer interface. The altered substrate specificity is a direct consequence of these substitutions.

Porcine Carbonyl Reductase STRUCTURAL BASIS FOR A FUNCTIONAL MONOMER IN SHORT CHAIN DEHYDROGENASES/REDUCTASES

Porcine testicular carbonyl reductase (PTCR) belongs to the short chain dehydrogenases/reductases (SDR) superfamily and catalyzes the NADPH-dependent reduction of ketones on steroids and prostaglandins. The enzyme shares nearly 85% sequence identity with the NADPH-dependent human 15-hydroxyprostaglandin dehydrogenase/carbonyl reductase. The tertiary structure of the enzyme at 2.3 Å reveals a fold characteristic of the SDR superfamily that uses a Tyr-Lys-Ser triad as catalytic residues, but exhibits neither the functional homotetramer nor the homodimer that distinguish all SDRs. It is the first known monomeric structure in the SDR superfamily. In PTCR, which is also active as a monomer, a 41-residue insertion immediately before the catalytic Tyr describes an all-helix subdomain that packs against interfacial helices, eliminating the four-helix bundle interface conserved in the superfamily. An additional anti-parallel strand in the PTCR structure also blocks the other strand-mediated interface. These novel structural features provide the basis for the scaffolding of one catalytic site within a single molecule of the enzyme.

Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed

KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. KillerRed could serve as an instrument to inactivate target proteins or to kill cell populations in photodynamic therapy. However, the nature of KillerRed phototoxicity has remained unclear, impeding the development of more phototoxic variants. Here we present the results of a high resolution crystallographic study of KillerRed in the active fluorescent and in the photobleached non-fluorescent states. A unique and striking feature of the structure is a water-filled channel reaching the chromophore area from the end cap of the β-barrel that is probably one of the key structural features responsible for phototoxicity. A study of the structure-function relationship of KillerRed, supported by structure-based, site-directed mutagenesis, has also revealed the key residues most likely responsible for the phototoxic effect. In particular, Glu68 and Ser119, located adjacent to the chromophore, have been assigned as the primary trigger of the reaction chain.

Steroid dehydrogenase structures, mechanism of action, and disease

Steroid dehydrogenase enzymes influence mammalian reproduction, hypertension, neoplasia, and digestion. The three-dimensional structures of steroid dehydrogenase enzymes reveal the position of the catalytic triad, a possible mechanism of keto-hydroxyl interconversion, a molecular mechanism of inhibition, and the basis for selectivity. Glycyrrhizic acid, the active ingredient in licorice, and its metabolite carbenoxolone are potent inhibitors of human 11 beta-hydroxysteroid dehydrogenase and bacterial 3 alpha, 20 beta-hydroxysteroid dehydrogenase (3 alpha, 20 beta-HSD). The three-dimensional structure of the 3 alpha, 20 beta-HSD carbenoxolone complex unequivocally verifies the postulated active site of the enzyme, shows that inhibition is a result of direct competition with the substrate for binding, and provides a plausible model for the mechanism of inhibition of 11 beta-hydroxysteroid dehydrogenase by carbenoxolone. The structure of the ternary complex of human 17 beta-hydroxysteroid dehydrogenase type 1 (17 beta-HSD) with the cofactor NADP+ and the antiestrogen equilin reveals the details of binding of an inhibitor in the active site of the enzyme and the possible roles of various amino acids in the catalytic cleft. The short-chain dehydrogenase reductase (SDR) family includes these steroid dehydrogenase enzymes and more than 60 other proteins from human, mammalian, insect, and bacterial sources. Most members of the family contain the tyrosine and lysine of the catalytic triad in a YxxxK sequence. X-ray crystal structures of 13 members of the family have been completed. When the alpha-carbon backbone of the cofactor binding domains of the structures are superimposed, the conserved residues are at the core of the structure and in the cofactor binding domain, but not in the substrate binding pocket.

Molecular structure and mechanisms of action of cyclic and linear ion transport antibiotics

Ionophores are antibiotics that induce ion transport across natural and artificial membranes. The specific function of a given ionophore depends upon its selectivity and the kinetics of ion capture, transport, and release. Systematic studies of complexed and uncomplexed forms of linear and cyclic ionophores provide insight into molecular mechanisms of ion capture and release and the basis for ion selectivity. The cyclic dodecadepsipeptide valinomycin, cyclo[(-L-Val-D-Hyi-D-Val-L-Lac)3-], transports potassium ions across cellular membrane bilayers selectively. The x-ray crystallographic and nmr spectroscopic data concerning the structures of Na+, K+, and Ba+2 complexes are consistent and provide a rationale for the K+ selectivity of valinomycin. Three significantly different conformations of valinomycin are observed in anhydrous crystals, in hydrated crystals grown from dimethylsulfoxide, and in crystals grown from dioxane. Each of these conformations suggests a different mechanism of ion capture. One of the observed conformations has an elliptical structure stabilized by four 4<--1 intramolecular hydrogen bonds and two 5<--1 hydrogen bonds. Ion capture could be readily achieved by disruption of the 5<--1 hydrogen bonds to permit coordination to a potassium ion entering the cavity. The conformation found in crystals obtained from dimethyl sulfoxide is an open flower shape having three petals and three 4<--1 hydrogen bonds. Complexation could proceed by a closing up of the three petals of the flower around the desolvating ion. In the third form, water molecules reside in the central cavity of a bracelet structure having six 4<--1 hydrogen bonds. Two of these bracelets stack over one another with their valine-rich faces surrounding a dioxane molecule. The stacked molecules form a channel approximately 20 A in length, suggesting that under certain circumstances valinomycin might function as a channel. A series of analogues of valinomycin differing in ring composition and size have been synthesized and their transport properties tested. Peptide substitution and chiral variation in the dodecadepsipeptide can result in stabilization or modification of the different conformers. While contraction of the ring size results in loss of ion transport properties, expansion of the ring size permits complexation of larger ions and small positively charged molecules. Gramicidin A is a pentadecapeptide that functions as a transmembrane channel for transporting monovalent cations. Crystal structures of the cesium chloride complex and two uncomplexed forms of gramicidin A have been reported. In all three structures the gramicidin A molecule is a left-handed, antiparallel, double-stranded helical dimer. In the cesium complex the beta 7.2-helix has 6.4 residues per turn with an internal cavity large enough to accommodate cesium ions. In the uncomplexed structures the channel is 31 A long and has 5.6 amino acids per turn. Because the helix is too tightly wound to permit ion transport, ion transport would require breaking and reforming of hydrogen bonds.

Rational proteomics I. Fingerprint identification and cofactor specificity in the short-chain oxidoreductase (SCOR) enzyme family

The short‐chain oxidoreductase (SCOR) family of enzymes includes over 2000 members identified in sequenced genomes. Of these enzymes, ∼200 have been characterized functionally, and the three‐dimensional crystal structures of ∼40 have been reported. Since some SCOR enzymes are involved in hypertension, diabetes, breast cancer, and polycystic kidney disease, it is important to characterize the other members of the family for which the biological functions are currently unknown. Although the SCOR family appears to have only a single fully conserved residue, it was possible, using bioinformatics methods, to determine characteristic fingerprints composed of 30–40 residues that are conserved at the 70% or greater level in SCOR subgroups. These fingerprints permit reliable prediction of several important structure‐function features including NAD/NADP cofactor preference. For example, the correlation of aspartate or arginine residues with NAD or NADP binding, respectively, predicts the cofactor preference of more than 70% of the SCOR proteins with unknown function. The analysis of conserved residues surrounding the cofactor has revealed the presence of previously undetected CH…O hydrogen bonds in the majority of the SCOR crystal structures, predicts the presence of similar hydrogen bonds in 90% of the SCOR proteins of unknown function, and suggests that these hydrogen bonds may play a critical role in the catalytic functions of these enzymes. Proteins 2003.

Crystal structure of bovine duodenase, a serine protease, with dual trypsin and chymotrypsin‐like specificities

The three‐dimensional structure of duodenase, a serine protease from bovine duodenum mucosa, has been determined at 2.4Å resolution. The enzyme, which has both trypsin‐like and chymotrypsin‐like activities, most closely resembles human cathepsin G with which it shares 57% sequence identity and similar specificity. The catalytic Ser195 in duodenase adopts the energetically favored conformation typical of serine proteinases and unlike the strained state typical of lipase/esterases. Of several waters in the active site of duodenase, the one associated with Ser214 is found in all serine proteinases and most lipase/esterases. The conservation of the Ser214 residue in serine proteinase, its presence in the active site, and participation in a hydrogen water network involving the catalytic triad (His57, Asp107, and Ser195) argues for its having an important role in the mechanism of action. It may be referred to as a fourth member of the catalytic triad. Duodenase is one of a growing family of enzymes that possesses trypsin‐like and chymotrypsin‐like activity. Not long ago, these activities were considered to be mutually exclusive. Computer modeling reveals that the S1 subsite of duodenase has structural features compatible with effective accommodation of P1 residues typical of trypsin (Arg/Lys) and chymotrypsin (Tyr/Phe) substrates. The determination of structural features associated with functional variation in the enzyme family may permit design of enzymes with a specific ratio of trypsin and chymotrypsin activities. Proteins 2000;41:8–16.

Rational proteomics IV: modeling the primary function of the mammalian 17β-hydroxysteroid dehydrogenase type 8

Significant sequence homology has been detected between prokaryotic beta-ketoacyl-[acyl carrier protein] reductases (BKR) and eukaryotic 17beta-hydroxysteroid dehydrogenases type 8 (17beta-HSD_8). Three-dimensional models of ternary complexes of human 17beta-HSD_8 with NAD cofactor and two chemically distinct substrates, the BKR substrate {CH3-(CH2)(12)-CO-CH(2)-CO-S-[ACP]} and the HSD substrate {estradiol} have been constructed (the atomic coordinates are available on request; e-mail: pletnev@hwi.buffalo.edu). The more extensive and specific interactions of 17beta-HSD_8 with the BKR substrate compared to interactions with estradiol raise a serious question about the enzymes primary function in vivo and suggest that it is likely to be involved in the regulation of fatty acid metabolism rather than in the steroid-dependent activity that has been demonstrated in vitro.