Crystal structure of pyruvate dehydrogenase phosphatase 1 and its functional implications

Pyruvate dehydrogenase phosphatase 1 (PDP1) catalyzes dephosphorylation of pyruvate dehydrogenase (E1) in the mammalian pyruvate dehydrogenase complex (PDC), whose activity is regulated by the phosphorylation-dephosphorylation cycle by the corresponding protein kinases (PDHKs) and phosphatases. The activity of PDP1 is greatly enhanced through Ca2+ -dependent binding of the catalytic subunit (PDP1c) to the L2 (inner lipoyl) domain of dihydrolipoyl acetyltransferase (E2), which is also integrated in PDC. Here, we report the crystal structure of the rat PDP1c at 1.8 A resolution. The structure reveals that PDP1 belongs to the PPM family of protein serine/threonine phosphatases, which, in spite of a low level of sequence identity, share the structural core consisting of the central beta-sandwich flanked on both sides by loops and alpha-helices. Consistent with the previous studies, two well-fixed magnesium ions are coordinated by five active site residues and five water molecules in the PDP1c catalytic center. Structural analysis indicates that, while the central portion of the PDP1c molecule is highly conserved among the members of the PPM protein family, a number of structural insertions and deletions located at the periphery of PDP1c likely define its functional specificity towards the PDC. One notable feature of PDP1c is a long insertion (residues 98-151) forming a unique hydrophobic pocket on the surface that likely accommodates the lipoyl moiety of the E2 domain in a fashion similar to that of PDHKs. The cavity, however, appears more open than in PDHK, suggesting that its closure may be required to achieve tight, specific binding of the lipoic acid. We propose a mechanism in which the closure of the lipoic acid binding site is triggered by the formation of the intermolecular (PDP1c/L2) Ca2+ binding site in a manner reminiscent of the Ca2+ -induced closure of the regulatory domain of troponin C.     

Crystal structure of human protein tyrosine phosphatase SHP-1 in the open conformation

SHP‐1 belongs to the family of non‐receptor protein tyrosine phosphatases (PTPs) and generally acts as a negative regulator in a variety of cellular signaling pathways. Previously, the crystal structures of the tail‐truncated SHP‐1 and SHP‐2 revealed an autoinhibitory conformation. To understand the regulatory mechanism of SHP‐1, we have determined the crystal structure of the full‐length SHP‐1 at 3.1 Å. Although the tail was disordered in current structure, the huge conformational rearrangement of the N‐SH2 domain and the incorporation of sulfate ions into the ligand‐binding site of each domain indicate that the SHP‐1 is in the open conformation. The N‐SH2 domain in current structure is shifted away from the active site of the PTP domain to the other side of the C‐SH2 domain, resulting in exposure of the active site. Meanwhile, the C‐SH2 domain is twisted anticlockwise by about 110°. In addition, a set of new interactions between two SH2 domains and between the N‐SH2 and the catalytic domains is identified, which could be responsible for the stabilization of SHP‐1 in the open conformation. Based on the structural comparison, a model for the activation of SHP‐1 is proposed. J. Cell. Biochem. 112: 2062–2071, 2011. © 2011 Wiley‐Liss, Inc.     

Crystal structure of human dipeptidyl peptidase IV/CD26 in complex with a substrate analog

Dipeptidyl peptidase IV (DPP-IV/CD26) is a multifunctional type II transmembrane serine peptidase. This enzyme contributes to the regulation of various physiological processes, including blood sugar homeostasis, by cleaving peptide hormones, chemokines and neuropeptides. We have determined the 2.5 A structure of the extracellular region of DPP-IV in complex with the inhibitor valine-pyrrolidide. The catalytic site is located in a large cavity formed between the alpha/beta-hydrolase domain and an eight-bladed beta-propeller domain. Both domains participate in inhibitor binding. The structure indicates how substrate specificity is achieved and reveals a new and unexpected opening to the active site.     

Steady-state kinetic analysis of human ubiquitin-activating enzyme (E1) using a fluorescently labeled ubiquitin substrate

We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys 6 of Ub. We demonstrate that Og-Ub is efficiently accepted by Ub-utilizing enzymes, such as the human ubiquitin-activating enzyme (E1). We used this fluorescent substrate to follow the steady-state kinetics of human E1-catalyzed Ub-transfer to the ubiquitin-carrier enzyme Ubc4. In this reaction, E1 uses three substrates: ATP, Ubc4, and Ub. The steady-state kinetics of Og-Ub utilization by E1 is presented. We have also used analytical ultracentrifugation methods to establish that E1 is monomeric under our assay condition (low salt) as well as under physiological condition (150 mM NaCl).     

Crystal structure of the heme d1 biosynthesis enzyme NirE in complex with its substrate reveals new insights into the catalytic mechanism of S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferases

During the biosynthesis of heme d₁, the essential cofactor of cytochrome cd₁ nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-l-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-l-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a “puckered” conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate.     

Crystal structure and substrate recognition mechanism of Aspergillus oryzae isoprimeverose-producing enzyme

Isoprimeverose-producing enzymes (IPases) release isoprimeverose (α-d-xylopyranosyl-(1?→?6)-d-glucopyranose) from the non-reducing end of xyloglucan oligosaccharides. Aspergillus oryzae IPase (IpeA) is classified as a member of the glycoside hydrolase family 3 (GH3); however, it has unusual substrate specificity compared with other GH3 enzymes. Xylopyranosyl branching at the non-reducing ends of xyloglucan oligosaccharides is vital for IpeA activity. We solved the crystal structure of IpeA with isoprimeverose at 2.4?Å resolution, showing that the structure of IpeA formed a dimer and was composed of three domains: an N-terminal (β/α)₈ TIM-barrel domain, α/β/α sandwich fold domain, and a C-terminal fibronectin-like domain. The catalytic TIM-barrel domain possessed a catalytic nucleophile (Asp300) and acid/base (Glu524) residues. Interestingly, we found that the cavity of the active site of IpeA was larger than that of other GH3 enzymes, and subsite ?1′ played an important role in its activity. The glucopyranosyl and xylopyranosyl residues of isoprimeverose were located at subsites ?1 and ?1′, respectively. Gln58 and Tyr89 contributed to the interaction with the xylopyranosyl residue of isoprimeverose through hydrogen bonding and stacking effects, respectively. Our findings provide new insights into the substrate recognition of GH3 enzymes.     

Crystal structure of the yeast Sac1: implications for its phosphoinositide phosphatase function

Sac family phosphoinositide (PI) phosphatases are an essential family of CX₅R(T/S)‐based enzymes, involved in numerous aspects of cellular function such as PI homeostasis, cellular signalling, and membrane trafficking. Genetic deletions of several Sac family members result in lethality in animal models and mutations of the Sac3 gene have been found in human hereditary diseases. In this study, we report the crystal structure of a founding member of this family, the Sac phosphatase domain of yeast Sac1. The 2.0 Å resolution structure shows that the Sac domain comprises of two closely packed sub‐domains, a novel N‐terminal sub‐domain and the PI phosphatase catalytic sub‐domain. The structure further shows a striking conformation of the catalytic P‐loop and a large positively charged groove at the catalytic site. These findings suggest an unusual mechanism for its dephosphorylation function. Homology structural modeling of human Fig4/Sac3 allows the mapping of several disease‐related mutations and provides a framework for the understanding of the molecular mechanisms of human diseases.     

Crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima

We have determined the crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima, TM0651 (gi 4981173), at 2.2 A resolution by selenomethionine single-wavelength anomalous diffraction (SAD) techniques. TM0651 is a member of the haloacid dehalogenase (HAD) superfamily, with sequence homology to trehalose-6-phosphate phosphatase and sucrose-6(F)-phosphate phosphohydrolase. Selenomethionine labeled TM0651 crystallized in space group C2 with three monomers per asymmetric unit. Each monomer has approximate dimensions of 65 x 40 x 35 A(3), and contains two domains: a domain of known hydrolase fold characteristic of the HAD family, and a domain with a new tertiary fold consisting of a six-stranded beta-sheet surrounded by four alpha-helices. There is one disulfide bond between residues Cys35 and Cys265 in each monomer. One magnesium ion and one sulfate ion are bound in the active site. The superposition of active site residues with other HAD family members indicates that TM0651 is very likely a phosphatase that acts through the formation of a phosphoaspartate intermediate, which is supported by both NMR titration data and a biochemical assay. Structural and functional database searches and the presence of many aromatic residues in the interface of the two domains suggest the substrate of TM0651 is a carbohydrate molecule. From the crystal structure and NMR data, the protein likely undergoes a conformational change upon substrate binding.     

Crystal structure of human TMDP, a testis-specific dual specificity protein phosphatase: implications for substrate specificity

The testis- and skeletal-muscle-specific dual-specificity phosphatase (TMDP) is a member of the dual-specificity phosphatase (DSP) subgroup of protein tyrosine phosphatases. TMDP has similar activities toward both tyrosine and threonine phosphorylated substrates, and is supposed to be involved in spermatogenesis. Here, we report the crystal structure of human TMDP at a resolution of 2.4 A. In spite of high sequence similarity with other DSPs, the crystal structure of TMDP shows distinct structural motifs and surface properties. In TMDP, the alpha1-beta1 loop, a substrate recognition motif is located further away from the active site loop in comparison to prototype DSP Vaccinia H1 related phophatase (VHR), which preferentially dephosphorylates tyrosine phosphorylated substrates and down-regulates MAP kinase signaling. Residues in the active site residues of TMDP are smaller in size and more hydrophobic than those of VHR. In addition, TMDP cannot be aligned with VHR in loop beta3-alpha4. These differences in the active site of TMDP result in a flat and wide pocket structure, allowing equal binding of phosphotyrosine and phosphothreonine substrates.     

Crystal structure of Ufc1, the Ufm1-conjugating enzyme

Ubiquitin and ubiquitin-like protein-conjugating enzymes play central roles in posttranslational modification processes. The ubiquitin-fold modifier 1 (Ufm1), one of a variety of ubiquitin-like modifiers, is covalently attached to target proteins via Uba5 and Ufm1-conjugating enzyme 1 (Ufc1), which are analogous to the E1 and E2 ubiquitylation enzymes. As Ufm1-related proteins are conserved in metazoa and plants, the Ufm1 system likely plays important roles in various multicellular organisms. Herein, we report the X-ray structure of human Ufc1 determined at 1.6 A resolution. The Ufc1 structure comprises a canonical E2 domain and an additional N-terminal domain. The Uba5 binding site on Ufc1 was assigned by structural comparison of Ufc1 and Ubc12 and related mutational analyses. In addition, we show that the N-terminal unique domain of Ufc1 contributes to thermal stability.     

Crystal structure of a minimal eIF4E-Cup complex reveals a general mechanism of eIF4E regulation in translational repression

Cup is an eIF4E-binding protein (4E-BP) that plays a central role in translational regulation of localized mRNAs during early Drosophila development. In particular, Cup is required for repressing translation of the maternally contributed oskar, nanos, and gurken mRNAs, all of which are essential for embryonic body axis determination. Here, we present the 2.8 Å resolution crystal structure of a minimal eIF4E–Cup assembly, consisting of the interacting regions of the two proteins. In the structure, two separate segments of Cup contact two orthogonal faces of eIF4E. The eIF4E-binding consensus motif of Cup (YXXXXLΦ) binds the convex side of eIF4E similarly to the consensus of other eIF4E-binding proteins, such as 4E-BPs and eIF4G. The second, noncanonical, eIF4E-binding site of Cup binds laterally and perpendicularly to the eIF4E β-sheet. Mutations of Cup at this binding site were shown to reduce binding to eIF4E and to promote the destabilization of the associated mRNA. Comparison with the binding mode of eIF4G to eIF4E suggests that Cup and eIF4G binding would be mutually exclusive at both binding sites. This shows how a common molecular surface of eIF4E might recognize different proteins acting at different times in the same pathway. The structure provides insight into the mechanism by which Cup disrupts eIF4E–eIF4G interaction and has broader implications for understanding the role of 4E-BPs in translational regulation.     

Crystal structure of the human dual specificity phosphatase 1 catalytic domain

The dual specificity phosphatase DUSP1 was the first mitogen activated protein kinase phosphatase (MKP) to be identified. It dephosphorylates conserved tyrosine and threonine residues in the activation loops of mitogen activated protein kinases ERK2, JNK1 and p38‐alpha. Here, we report the crystal structure of the human DUSP1 catalytic domain at 2.49 Å resolution. Uniquely, the protein was crystallized as an MBP fusion protein in complex with a monobody that binds to MBP. Sulfate ions occupy the phosphotyrosine and putative phosphothreonine binding sites in the DUSP1 catalytic domain.     

Crystal structure of human estrogen-related receptor alpha in complex with a synthetic inverse agonist reveals its novel molecular mechanism

Inverse agonists of the constitutively active human estrogen-related receptor alpha (ERRalpha, NR3B1) are of potential interest for several disease indications (e.g. breast cancer, metabolic diseases, or osteoporosis). ERRalpha is constitutively active, because its ligand binding pocket (LBP) is practically filled with side chains (in particular with Phe(328), which is replaced by Ala in ERRbeta and ERRgamma). We present here the crystal structure of the ligand binding domain of ERRalpha (containing the mutation C325S) in complex with the inverse agonist cyclohexylmethyl-(1-p-tolyl-1H-indol-3-ylmethyl)-amine (compound 1a), to a resolution of 2.3A(.) The structure reveals the dramatic multiple conformational changes in the LBP, which create the necessary space for the ligand. As a consequence of the new side chain conformation of Phe(328) (on helix H3), Phe(510)(H12) has to move away, and thus the activation helix H12 is displaced from its agonist position. This is a novel mechanism of H12 inactivation, different from ERRgamma, estrogen receptor (ER) alpha, and ERbeta. H12 binds (with a surprising binding mode) in the coactivator groove of its ligand binding domain, at a similar place as a coactivator peptide. This is in contrast to ERRgamma but resembles the situation for ERalpha (raloxifene or 4-hydroxytamoxifen complexes). Our results explain the novel molecular mechanism of an inverse agonist for ERRalpha and provide the basis for rational drug design to obtain isotype-specific inverse agonists of this potential new drug target. Despite a practically filled LBP, the finding that a suitable ligand can induce an opening of the cavity also has broad implications for other orphan nuclear hormone receptors (e.g. the NGFI-B subfamily).     

1H, 13C and 15N resonance assignments for the human E2 conjugating enzyme, UbcH7

UbcH7 is a human E2 conjugating enzyme in the ubiquitin-dependent protein degradation pathway. The resonance assignments of UbcH7 will assist in elucidating the structural basis of interactions that occur within ubiquitination.     

Crystal structure of human glyoxalase II and its complex with a glutathione thiolester substrate analogue.

BACKGROUND: Glyoxalase II, the second of two enzymes in the glyoxalase system, is a thiolesterase that catalyses the hydrolysis of S-D-lactoylglutathione to form glutathione and D-lactic acid. RESULTS: The structure of human glyoxalase II was solved initially by single isomorphous replacement with anomalous scattering and refined at a resolution of 1.9 A. The enzyme consists of two domains. The first domain folds into a four-layered beta sandwich, similar to that seen in the metallo-beta-lactamases. The second domain is predominantly alpha-helical. The active site contains a binuclear zinc-binding site and a substrate-binding site extending over the domain interface. The model contains acetate and cacodylate in the active site. A second complex was derived from crystals soaked in a solution containing the slow substrate, S-(N-hydroxy-N-bromophenylcarbamoyl)glutathione. This complex was refined at a resolution of 1.45 A. It contains the added ligand in one molecule of the asymmetric unit and glutathione in the other. CONCLUSIONS: The arrangement of ligands around the zinc ions includes a water molecule, presumably in the form of a hydroxide ion, coordinated to both metal ions. This hydroxide ion is situated 2.9 A from the carbonyl carbon of the substrate in such a position that it could act as the nucleophile during catalysis. The reaction mechanism may also have implications for the action of metallo-beta-lactamases.