Youzhong Guo

Protein structure. Structure and activity of tryptophan-rich TSPO proteins.

Structural clues to protein function Translocator protein (TSPO) is a mitochondrial membrane protein thought to transport cholesterol and porphyrins. Its detailed function remains unclear, but interest in it is high because TSPO is involved in a variety of human diseases. Two papers now present crystal structures of bacterial TSPOs. Li et al. show that a mutant that mimics a human single polymorphism associated with psychiatric disorders has structural changes in a region implicated in cholesterol binding. Guo et al. suggest that TSPO may be more than a transporter. They show how it catalyzes the degradation of porphyrins, a function that could be important in protection against oxidative stress. Science, this issue p. 555, p. 551 Structures of bacterial homologs give insight into TSPO function in human diseases. Translocator proteins (TSPOs) bind steroids and porphyrins, and they are implicated in many human diseases, for which they serve as biomarkers and therapeutic targets. TSPOs have tryptophan-rich sequences that are highly conserved from bacteria to mammals. Here we report crystal structures for Bacillus cereus TSPO (BcTSPO) down to 1.7 Å resolution, including a complex with the benzodiazepine-like inhibitor PK11195. We also describe BcTSPO-mediated protoporphyrin IX (PpIX) reactions, including catalytic degradation to a previously undescribed heme derivative. We used structure-inspired mutations to investigate reaction mechanisms, and we showed that TSPOs from Xenopus and man have similar PpIX-directed activities. Although TSPOs have been regarded as transporters, the catalytic activity in PpIX degradation suggests physiological importance for TSPOs in protection against oxidative stress.

Structure and selectivity in bestrophin ion channels.

Human bestrophin-1 (hBest1) is a calcium-activated chloride channel from the retinal pigment epithelium, where mutations are associated with vitelliform macular degeneration, or Best disease. We describe the structure of a bacterial homolog (KpBest) of hBest1 and functional characterizations of both channels. KpBest is a pentamer that forms a five-helix transmembrane pore, closed by three rings of conserved hydrophobic residues, and has a cytoplasmic cavern with a restricted exit. From electrophysiological analysis of structure-inspired mutations in KpBest and hBest1, we find a sensitive control of ion selectivity in the bestrophins, including reversal of anion/cation selectivity, and dramatic activation by mutations at the cytoplasmic exit. A homology model of hBest1 shows the locations of disease-causing mutations and suggests possible roles in regulation. A bacterial homolog structure gives insights into ion permeation, gating, and mutations that cause retinal degeneration. Insight into a retinal degeneration disease Human bestrophin 1 (hBest1) is a membrane protein that forms a chloride channel in the retinal pigment epithelium. Mutations in hBest1 can lead to a retinal degeneration disease known as Best disease. Yang et al. describe the structure of KpBest, a bacterial homolog of hBest1. KpBest forms a pentamer with an ion channel at its center. In contrast to hBest1, KpBest1 is a sodium channel. The structure suggests a mechanism for ion selectivity that was confirmed by mutagenesis of KpBest and hBest1. A model of the hBest1 channel structure based on the KpBest structure reveals how mutations cause disease. Science, this issue p. 355

Structure and activity of tryptophan-rich TSPO proteins

Structural clues to protein function Translocator protein (TSPO) is a mitochondrial membrane protein thought to transport cholesterol and porphyrins. Its detailed function remains unclear, but interest in it is high because TSPO is involved in a variety of human diseases. Two papers now present crystal structures of bacterial TSPOs. Li et al. show that a mutant that mimics a human single polymorphism associated with psychiatric disorders has structural changes in a region implicated in cholesterol binding. Guo et al. suggest that TSPO may be more than a transporter. They show how it catalyzes the degradation of porphyrins, a function that could be important in protection against oxidative stress. Science, this issue p. 555, p. 551 Structures of bacterial homologs give insight into TSPO function in human diseases. Translocator proteins (TSPOs) bind steroids and porphyrins, and they are implicated in many human diseases, for which they serve as biomarkers and therapeutic targets. TSPOs have tryptophan-rich sequences that are highly conserved from bacteria to mammals. Here we report crystal structures for Bacillus cereus TSPO (BcTSPO) down to 1.7 Å resolution, including a complex with the benzodiazepine-like inhibitor PK11195. We also describe BcTSPO-mediated protoporphyrin IX (PpIX) reactions, including catalytic degradation to a previously undescribed heme derivative. We used structure-inspired mutations to investigate reaction mechanisms, and we showed that TSPOs from Xenopus and man have similar PpIX-directed activities. Although TSPOs have been regarded as transporters, the catalytic activity in PpIX degradation suggests physiological importance for TSPOs in protection against oxidative stress.

Multi-crystal native SAD analysis at 6 keV.

Anomalous diffraction signals from typical native macromolecules are very weak, frustrating their use in de novo structure determination. Here, native SAD procedures are described to enhance signal to noise in anomalous diffraction by using multiple crystals in combination with synchrotron X-rays at 6 keV. Increased anomalous signals were obtained at 6 keV compared with 7 keV X-ray energy, which was used for previous native SAD analyses. A feasibility test of multi-crystal-based native SAD phasing was performed at 3.2 Å resolution for a known tyrosine protein kinase domain, and real-life applications were made to two novel membrane proteins at about 3.0 Å resolution. The three applications collectively serve to validate the robust feasibility of native SAD phasing at lower energy.

Crystal structures of native and inactivated cis-3-chloroacrylic acid dehalogenase: Implications for the catalytic and inactivation mechanisms.

The isomeric mixture of cis- and trans-1,3-dichloropropene constitutes the active component of a widely used nematocide known as Telone II®. The mixture is processed by various soil bacteria to acetaldehyde through the 1,3-dichloropropene catabolic pathway. The pathway relies on an isomer-specific hydrolytic dehalogenation reaction catalyzed by cis- or trans-3-chloroacrylic acid dehalogenase, known respectively as cis-CaaD and CaaD. Previous sequence analysis and crystallographic studies of the native and covalently modified enzymes identified Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114 as key binding and catalytic residues in cis-CaaD. Mutagenesis of these residues confirmed their importance to the dehalogenation reaction. Crystal structures of the native enzyme (2.01Å resolution) and the enzyme covalently modified at the Pro-1 nitrogen by 2-hydroxypropanoate (1.65Å resolution) are reported here. Both structures are at a resolution higher than previously reported (2.75Å and 2.1Å resolution, respectively). The conformation of the covalent adduct is strikingly different from that previously reported due to its interaction with a 7-residue loop (Thr-32 to Leu-38). The participation of another active site residue, Arg-117, in catalysis and inactivation was also examined. The implications of the combined findings for the mechanisms of catalysis and inactivation are discussed.

A Kazal-Type Serine Protease Inhibitor from the Defense Gland Secretion of the Subterranean Termite Coptotermes formosanus Shiraki.

Coptotermes formosanus is an imported, subterranean termite species with the largest economic impact in the United States. The frontal glands of the soldier caste termites comprising one third of the body mass, contain a secretion expelled through a foramen in defense. The small molecule composition of the frontal gland secretion is well-characterized, but the proteins remain to be identified. Herein is reported the structure and function of one of several proteins found in the termite defense gland secretion. TFP4 is a 6.9 kDa, non-classical group 1 Kazal-type serine protease inhibitor with activity towards chymotrypsin and elastase, but not trypsin. The 3-dimensional solution structure of TFP4 was solved with nuclear magnetic resonance spectroscopy, and represents the first structure from the taxonomic family, Rhinotermitidae. Based on the structure of TFP4, the protease inhibitor active loop (Cys8 to Cys16) was identified.

Kinetic, Mutational, and Structural Analysis of Malonate Semialdehyde Decarboxylase from Coryneform Bacterium Strain FG41: Mechanistic Implications for the Decarboxylase and Hydratase Activities.

Malonate semialdehyde decarboxylase from Pseudomonas pavonaceae 170 (designated Pp MSAD) is in a bacterial catabolic pathway for the nematicide 1,3-dichloropropene. MSAD has two known activities: it catalyzes the metal ion-independent decarboxylation of malonate semialdehyde to produce acetaldehyde and carbon dioxide and a low-level hydration of 2-oxo-3-pentynoate to yield acetopyruvate. The latter activity is not known to be biologically relevant. Previous studies identified Pro-1, Asp-37, and a pair of arginines (Arg-73 and Arg-75) as critical residues in these activities. In terms of pairwise sequence, MSAD from Coryneform bacterium strain FG41 (designated FG41 MSAD) is 38% identical with the Pseudomonas enzyme, including Pro-1 and Asp-37. However, Gln-73 replaces Arg-73, and the second arginine is shifted to Arg-76 by the insertion of a glycine. To determine how these changes relate to the activities of FG41 MSAD, the gene was cloned and the enzyme expressed and characterized. The enzyme has a comparable decarboxylase activity but a significantly reduced hydratase activity. Mutagenesis along with crystal structures of the native enzyme (2.0 Å resolution) and the enzyme modified by a 3-oxopropanoate moiety (resulting from the incubation of the enzyme and 3-bromopropiolate) (2.2 Å resolution) provided a structural basis. The roles of Pro-1 and Asp-37 are likely the same as those proposed for Pp MSAD. However, the side chains of Thr-72, Gln-73, and Tyr-123 replace those of Arg-73 and Arg-75 in the mechanism and play a role in binding and catalysis. The structures also show that Arg-76 is likely too distant to play a direct role in the mechanism. FG41 MSAD is the second functionally annotated homologue in the MSAD family of the tautomerase superfamily and could represent a new subfamily.

Structure and Activity of Tryptophan-rich TSPO Translocator Proteins

Structural clues to protein function Translocator protein (TSPO) is a mitochondrial membrane protein thought to transport cholesterol and porphyrins. Its detailed function remains unclear, but interest in it is high because TSPO is involved in a variety of human diseases. Two papers now present crystal structures of bacterial TSPOs. Li et al. show that a mutant that mimics a human single polymorphism associated with psychiatric disorders has structural changes in a region implicated in cholesterol binding. Guo et al. suggest that TSPO may be more than a transporter. They show how it catalyzes the degradation of porphyrins, a function that could be important in protection against oxidative stress. Science, this issue p. 555, p. 551 Structures of bacterial homologs give insight into TSPO function in human diseases. Translocator proteins (TSPOs) bind steroids and porphyrins, and they are implicated in many human diseases, for which they serve as biomarkers and therapeutic targets. TSPOs have tryptophan-rich sequences that are highly conserved from bacteria to mammals. Here we report crystal structures for Bacillus cereus TSPO (BcTSPO) down to 1.7 Å resolution, including a complex with the benzodiazepine-like inhibitor PK11195. We also describe BcTSPO-mediated protoporphyrin IX (PpIX) reactions, including catalytic degradation to a previously undescribed heme derivative. We used structure-inspired mutations to investigate reaction mechanisms, and we showed that TSPOs from Xenopus and man have similar PpIX-directed activities. Although TSPOs have been regarded as transporters, the catalytic activity in PpIX degradation suggests physiological importance for TSPOs in protection against oxidative stress.

Comparison of the Structures and Activities of Cg10062 with other 4-OT Enzymes

The tautomerase superfamily is a broad family of proteins represented by 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2hydroxymuconate isomerase (CHMI), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), malonate semialdehyde decarboxylase (MSAD), and macrophage migration inhibitory factor (MIF). 4-OT and many of its homologues are homoor heterohexamers composed of small (60-75 a.a. residue) subunits while CHMI, MSAD, cis-CaaD and MIF are nearly twice that size and form trimers. The subunits of this family share two distinguishing features – one or two beta-alpha-beta structural motifs and a catalytically important N-terminal Pro residue. Several different catalytic activities are known to utilize this same structural motif tautomerase, isomerase, decarboxylase, dehalogenase, etc. Cg10062 is a homologue of cis-CaaD, however, it differs from cis-CaaD in several respects. In addition to being able to process cis-3-chloroacrylic acid, it also displays multiple other functions such as the capability to process trans-3-chloroacrylic acid (like CaaD), to process phenylpyruvate (like PPT/MIF) and 2-oxo-3-pentynoate (like CaaD, cis-CaaD and MSAD). Furthermore, Cg10062 is inactivated by both (R) and (S)-oxirane-2-carboxylate. However, cis-CaaD is only inactivated by the (R)isomer. The X-ray structures of native Cg10062, and its inactivated complexes have been determined and the results of comparing these structures and activities with CaaD, cis-CaaD, PPT/MIF and MSAD will be reported. This work was supported in part by The Welch Foundation ( F1334).