Crystal structures of the myristylated catalytic subunit of cAMP-dependent protein kinase reveal open and closed conformations.

Three crystal structures, representing two distinct conformational states, of the mammalian catalytic subunit of cAMP-dependent protein kinase were solved using molecular replacement methods starting from the refined structure of the recombinant catalytic subunit ternary complex (Zheng, J., et al., 1993a, Biochemistry 32, 2154-2161). These structures correspond to the free apoenzyme, a binary complex with an iodinated inhibitor peptide, and a ternary complex with both ATP and the unmodified inhibitor peptide. The apoenzyme and the binary complex crystallized in an open conformation, whereas the ternary complex crystallized in a closed conformation similar to the ternary complex of the recombinant enzyme. The model of the binary complex, refined at 2.9 A resolution, shows the conformational changes associated with the open conformation. These can be described by a rotation of the small lobe and a displacement of the C-terminal 30 residues. This rotation of the small lobe alters the cleft interface in the active-site region surrounding the glycine-rich loop and Thr 197, a critical phosphorylation site. In addition to the conformational changes, the myristylation site, absent in the recombinant enzyme, was clearly defined in the binary complex. The myristic acid binds in a deep hydrophobic pocket formed by four segments of the protein that are widely dispersed in the linear sequence. The N-terminal 40 residues that lie outside the conserved catalytic core are anchored by the N-terminal myristylate plus an amphipathic helix that spans both lobes and is capped by Trp 30. Both posttranslational modifications, phosphorylation and myristylation, contribute directly to the stable structure of this enzyme.     

Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism

BACKGROUND: The bacterial heat shock locus HslU ATPase and HslV peptidase together form an ATP-dependent HslVU protease. Bacterial HslVU is a homolog of the eukaryotic 26S proteasome. Crystallographic studies of HslVU should provide an understanding of ATP-dependent protein unfolding, translocation, and proteolysis by this and other ATP-dependent proteases. RESULTS: We present a 3.0 A resolution crystal structure of HslVU with an HslU hexamer bound at one end of an HslV dodecamer. The structure shows that the central pores of the ATPase and peptidase are next to each other and aligned. The central pore of HslU consists of a GYVG motif, which is conserved among protease-associated ATPases. The binding of one HslU hexamer to one end of an HslV dodecamer in the 3.0 A resolution structure opens both HslV central pores and induces asymmetric changes in HslV. CONCLUSIONS: Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.     

Crystal structures of a family 8 polysaccharide lyase reveal open and highly occluded substrate-binding cleft conformations

Bacterial enzymatic degradation of glycosaminoglycans such as hyaluronan and chondroitin is facilitated by polysaccharide lyases. Family 8 polysaccharide lyase (PL8) enzymes contain at least two domains: one predominantly composed of α-helices, the α-domain, and another predominantly composed of β-sheets, the β-domain. Simulation flexibility analyses indicate that processive exolytic cleavage of hyaluronan, by PL8 hyaluronate lyases, is likely to involve an interdomain shift, resulting in the opening/closing of the substrate-binding cleft between the α- and β-domains, facilitating substrate translocation. Here, the Streptomyces coelicolor A3(2) PL8 enzyme was recombinantly expressed in and purified from Escherichia coli and biochemically characterized as a hyaluronate lyase. By using X-ray crystallography its structure was solved in complex with hyaluronan and chondroitin disaccharides. These findings show key catalytic interactions made by the different substrates, and on comparison with all other PL8 structures reveals that the substrate-binding cleft of the S. coelicolor enzyme is highly occluded. A third structure of the enzyme, harboring a mutation of the catalytic tyrosine, created via site-directed mutagenesis, interestingly revealed an interdomain shift that resulted in the opening of the substrate-binding cleft. These results add further support to the proposed processive mechanism of action of PL8 hyaluronate lyases and may indicate that the mechanism of action is likely to be universally used by PL8 hyaluronate lyases.     

Crystal structures of c-Src reveal features of its autoinhibitory mechanism.

Src family kinases are maintained in an assembled, inactive conformation by intramolecular interactions of their SH2 and SH3 domains. Full catalytic activity requires release of these restraints as well as phosphorylation of Tyr-416 in the activation loop. In previous structures of inactive Src kinases, Tyr-416 and flanking residues are disordered. We report here four additional c-Src structures in which this segment adopts an ordered but inhibitory conformation. The ordered activation loop forms an alpha helix that stabilizes the inactive conformation of the kinase domain, blocks the peptide substrate-binding site, and prevents Tyr-416 phosphorylation. Disassembly of the regulatory domains, induced by SH2 or SH3 ligands, or by dephosphorylation of Tyr-527, could lead to exposure and phosphorylation of Tyr-416.     

Crystal structures of manganese- and cobalt-substituted myoglobin in complex with NO and nitrite reveal unusual ligand conformations

Nitrite is now recognized as a storage pool of bioactive nitric oxide (NO). Hemoglobin (Hb) and myoglobin (Mb) convert, under certain conditions, nitrite to NO. This newly discovered nitrite reductase activity of Hb and Mb provides an attractive alternative to mammalian NO synthesis from the NO synthase pathway that requires dioxygen. We recently reported the X-ray crystal structure of the nitrite adduct of ferric horse heart Mb, and showed that the nitrite ligand binds in an unprecedented O-binding (nitrito) mode to the d(5) ferric center in Mb(III)(ONO) [D.M. Copeland, A. Soares, A.H. West, G.B. Richter-Addo, J. Inorg. Biochem. 100 (2006) 1413-1425]. We also showed that the distal pocket in Mb allows for different conformations of the NO ligand (120 degrees and 144 degrees ) in Mb(II)NO depending on the mode of preparation of the compound. In this article, we report the crystal structures of the nitrite and NO adducts of manganese-substituted hh Mb (a d(4) system) and of the nitrite adduct of cobalt-substituted hh Mb (a d(6) system). We show that the distal His64 residue directs the nitrite ligand towards the rare nitrito O-binding mode in Mn(III)Mb and Co(III)Mb. We also report that the distal pocket residues allow a stabilization of an unprecendented bent MnNO moiety in Mn(II)MbNO. These crystal structural data, when combined with the data for the aquo, methanol, and azide MnMb derivatives, provide information on the role of distal pocket residues in the observed binding modes of nitrite and NO ligands to wild-type and metal-substituted Mb.     

Crystal and solution structures of a superantigen from Yersinia pseudotuberculosis reveal a jelly-roll fold

Superantigens are a class of microbial proteins with the ability to excessively activate T cells by binding to the T cell receptor. The staphylococcal and streptococcal superantigens are closely related in structure and possess an N-terminal domain that resembles an OB fold and a C-terminal domain similar to a beta-grasp fold. Yersinia pseudotuberculosis produces superantigens, YPMa, YPMb, and YPMc, which have no significant amino acid similarity to other proteins. We have determined the crystal and solution structures of YPMa, which show that the protein has a jelly-roll fold. The closest structural neighbors to YPMa are viral capsid proteins and members of the tumor necrosis factor superfamily. In the crystal structure, YPMa packs as a trimer, another feature shared with viral capsid proteins and TNF superfamily proteins. However, in solution YPMa behaves as a monomer, and any functional relevance of the trimer observed in the crystals is yet to be established.     

Crystal structures of RNase H2 in complex with nucleic acid reveal the mechanism of RNA-DNA junction recognition and cleavage

Two classes of RNase H hydrolyze RNA of RNA/DNA hybrids. In contrast to RNase H1 that requires four ribonucleotides for cleavage, RNase H2 can nick duplex DNAs containing a single ribonucleotide, suggesting different in vivo substrates. We report here the crystal structures of a type 2 RNase H in complex with substrates containing a (5')RNA-DNA(3') junction. They revealed a unique mechanism of recognition and substrate-assisted cleavage. A conserved tyrosine residue distorts the nucleic acid at the junction, allowing the substrate to function in catalysis by participating in coordination of the active site metal ion. The biochemical and structural properties of RNase H2 explain the preference of the enzyme for junction substrates and establish the structural and mechanistic differences with RNase H1. Junction recognition is important for the removal of RNA embedded in DNA and may play an important role in DNA replication and repair.     

Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus reveal two conformations and suggest mechanisms of inhibition by non-nucleoside inhibitors

Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus (HCV) from two crystal forms have been determined. Similar to the three-dimensional structures of HCV polymerase genotype 1b and other known polymerases, the structures of the HCV polymerase genotype 2a in both crystal forms can be depicted in the classical right-hand arrangement with fingers, palm, and thumb domains. The main structural differences between the molecules in the two crystal forms lie at the interface of the fingers and thumb domains. The relative orientation of the thumb domain with respect to the fingers and palm domains and the beta-flap region is altered. Structural analysis reveals that the NS5B polymerase in crystal form I adopts a "closed" conformation that is believed to be the active form, whereas NS5B in crystal form II adopts an "open" conformation and is thus in the inactive form. In addition, we have determined the structures of two NS5B polymerase/non-nucleoside inhibitor complexes. Both inhibitors bind at a common binding site, which is nearly 35 A away from the polymerase active site and is located in the thumb domain. The binding pocket is predominantly hydrophobic in nature, and the enzyme inhibitor complexes are stabilized by hydrogen bonding and van der Waals interactions. Inhibitors can only be soaked in crystal form I and not in form II; examination of the enzyme-inhibitor complex reveals that the enzyme has undergone a dramatic conformational change from the form I (active) complex to the form II (inactive).     

Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism

Creatininase from Pseudomonas putida is a member of the urease-related amidohydrolase superfamily. The crystal structure of the Mn-activated enzyme has been solved by the single isomorphous replacement method at 1.8A resolution. The structures of the native creatininase and the Mn-activated creatininase-creatine complex have been determined by a difference Fourier method at 1.85 A and 1.6 A resolution, respectively. We found the disc-shaped hexamer to be roughly 100 A in diameter and 50 A in thickness and arranged as a trimer of dimers with 32 (D3) point group symmetry. The enzyme is a typical Zn2+ enzyme with a binuclear metal center (metal1 and metal2). Atomic absorption spectrometry and X-ray crystallography revealed that Zn2+ at metal1 (Zn1) was easily replaced with Mn2+ (Mn1). In the case of the Mn-activated enzyme, metal1 (Mn1) has a square-pyramidal geometry bound to three protein ligands of Glu34, Asp45, and His120 and two water molecules. Metal2 (Zn2) has a well-ordered tetrahedral geometry bound to the three protein ligands of His36, Asp45, and Glu183 and a water molecule. The crystal structure of the Mn-activated creatininase-creatine complex, which is the first structure as the enzyme-substrate/inhibitor complex of creatininase, reveals that significant conformation changes occur at the flap (between the alpha5 helix and the alpha6 helix) of the active site and the creatine is accommodated in a hydrophobic pocket consisting of Trp174, Trp154, Tyr121, Phe182, Tyr153, and Gly119. The high-resolution crystal structure of the creatininase-creatine complex enables us to identify two water molecules (Wat1 and Wat2) that are possibly essential for the catalytic mechanism of the enzyme. The structure and proposed catalytic mechanism of the creatininase are different from those of urease-related amidohydrolase superfamily enzymes. We propose a new two-step catalytic mechanism possibly common to creatininases in which the Wat1 acts as the attacking nucleophile in the water-adding step and the Wat2 acts as the catalytic acid in the ring-opening step.     

Crystal structures of bacterial peptidoglycan amidase AmpD and an unprecedented activation mechanism

AmpD is a cytoplasmic peptidoglycan (PG) amidase involved in bacterial cell-wall recycling and in induction of β-lactamase, a key enzyme of β-lactam antibiotic resistance. AmpD belongs to the amidase_2 family that includes zinc-dependent amidases and the peptidoglycan-recognition proteins (PGRPs), highly conserved pattern-recognition molecules of the immune system. Crystal structures of Citrobacter freundii AmpD were solved in this study for the apoenzyme, for the holoenzyme at two different pH values, and for the complex with the reaction products, providing insights into the PG recognition and the catalytic process. These structures are significantly different compared with the previously reported NMR structure for the same protein. The NMR structure does not possess an accessible active site and shows the protein in what is proposed herein as an inactive “closed” conformation. The transition of the protein from this inactive conformation to the active “open” conformation, as seen in the x-ray structures, was studied by targeted molecular dynamics simulations, which revealed large conformational rearrangements (as much as 17 Å) in four specific regions representing one-third of the entire protein. It is proposed that the large conformational change that would take the inactive NMR structure to the active x-ray structure represents an unprecedented mechanism for activation of AmpD. Analysis is presented to argue that this activation mechanism might be representative of a regulatory process for other intracellular members of the bacterial amidase_2 family of enzymes.     

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gamma phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.     

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gi phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.     

Crystal structures of human caspase 6 reveal a new mechanism for intramolecular cleavage self-activation

Dimeric effectors caspase 3 and caspase 7 are activated by initiator caspase processing. In this study, we report the crystal structures of effector caspase 6 (CASP6) zymogen and N-Acetyl-Val-Glu-Ile-Asp-al-inhibited CASP6. Both of these forms of CASP6 have a dimeric structure, and in CASP6 zymogen the intersubunit cleavage site (190)TEVD(193) is well structured and inserts into the active site. This positions residue Asp 193 to be easily attacked by the catalytic residue Cys 163. We demonstrate biochemically that intramolecular cleavage at Asp 193 is a prerequisite for CASP6 self-activation and that this activation mechanism is dependent on the length of the L2 loop. Our results indicate that CASP6 can be activated and regulated through intramolecular self-cleavage.     

Crystal structures of the Cid1 poly (U) polymerase reveal the mechanism for UTP selectivity

Polyuridylation is emerging as a ubiquitous post-translational modification with important roles in multiple aspects of RNA metabolism. These poly (U) tails are added by poly (U) polymerases with homology to poly (A) polymerases; nevertheless, the selection for UTP over ATP remains enigmatic. We report the structures of poly (U) polymerase Cid1 from Schizoscaccharomyces pombe alone and in complex with UTP, CTP, GTP and 3′-dATP. These structures reveal that each of the 4 nt can be accommodated at the active site; however, differences exist that suggest how the polymerase selects UTP over the other nucleotides. Furthermore, we find that Cid1 shares a number of common UTP recognition features with the kinetoplastid terminal uridyltransferases. Kinetic analysis of Cid1’s activity for its preferred substrates, UTP and ATP, reveal a clear preference for UTP over ATP. Ultimately, we show that a single histidine in the active site plays a pivotal role for poly (U) activity. Notably, this residue is typically replaced by an asparagine residue in Cid1-family poly (A) polymerases. By mutating this histidine to an asparagine residue in Cid1, we diminished Cid1’s activity for UTP addition and improved ATP incorporation, supporting that this residue is important for UTP selectivity.     

Crystal and electron microscopy structures of sticholysin II actinoporin reveal insights into the mechanism of membrane pore formation

Sticholysin II (StnII) is a pore-forming protein (PFP) produced by the sea anemone Stichodactyla helianthus. We found out that StnII exists in a monomeric soluble state but forms tetramers in the presence of a lipidic interface. Both structures have been independently determined at 1.7 A and 18 A resolution, respectively, by using X-ray crystallography and electron microscopy of two-dimensional crystals. Besides, the structure of soluble StnII complexed with phosphocholine, determined at 2.4 A resolution, reveals a phospholipid headgroup binding site, which is located in a region with an unusually high abundance of aromatic residues. Fitting of the atomic model into the electron microscopy density envelope suggests that while the beta sandwich structure of the protein remains intact upon oligomerization, the N-terminal region and a flexible and highly basic loop undergo significant conformational changes. These results provide the structural basis for the membrane recognition step of actinoporins and unexpected insights into the oligomerization step.     

Crystal structures of a Formin Homology-2 domain reveal a tethered dimer architecture

Formin proteins participate in a wide range of cytoskeletal processes in all eukaryotes. The defining feature of formins is a highly conserved approximately 400 residue region, the Formin Homology-2 (FH2) domain, which has recently been found to nucleate actin filaments. Here we report crystal structures of the S. cerevesiae Bni1p FH2 domain. The mostly alpha-helical FH2 domain forms a unique "tethered dimer" in which two elongated actin binding heads are tied together at either end by an unusual lasso and linker structure. Biochemical and crystallographic observations indicate that the dimer is stable but flexible, with flexibility between the two halves of the dimer conferred by the linker segments. Although each half of the dimer is competent to interact with filament ends, the intact dimer is required for actin nucleation and processive capping. The tethered dimer architecture may allow formins to stair-step on the barbed end of an elongating nascent filament.     

Crystal structures of penicillin-binding protein 2 from penicillin-susceptible and -resistant strains of Neisseria gonorrhoeae reveal an unexpectedly subtle mechanism for antibiotic resistance

Penicillin-binding protein 2 (PBP2) from N. gonorrhoeae is the major molecular target for beta-lactam antibiotics used to treat gonococcal infections. PBP2 from penicillin-resistant strains of N. gonorrhoeae harbors an aspartate insertion after position 345 (Asp-345a) and 4-8 additional mutations, but how these alter the architecture of the protein is unknown. We have determined the crystal structure of PBP2 derived from the penicillin-susceptible strain FA19, which shows that the likely effect of Asp-345a is to alter a hydrogen-bonding network involving Asp-346 and the SXN triad at the active site. We have also solved the crystal structure of PBP2 derived from the penicillin-resistant strain FA6140 that contains four mutations near the C terminus of the protein. Although these mutations lower the second order rate of acylation for penicillin by 5-fold relative to wild type, comparison of the two structures shows only minor structural differences, with the positions of the conserved residues in the active site essentially the same in both. Kinetic analyses indicate that two mutations, P551S and F504L, are mainly responsible for the decrease in acylation rate. Melting curves show that the four mutations lower the thermal stability of the enzyme. Overall, these data suggest that the molecular mechanism underlying antibiotic resistance contributed by the four mutations is subtle and involves a small but measurable disordering of residues in the active site region that either restricts the binding of antibiotic or impedes conformational changes that are required for acylation by beta-lactam antibiotics.     

Crystal structures of a Rab protein in its inactive and active conformations

We have determined crystal structures of Sec4, a member of the Rab family in the G protein superfamily, in two states: bound to GDP, and to a non-hydrolyzable GTP analog, guanosine-5'-(beta, gamma)-imidotriphosphate (GppNHp). This represents the first structure of a Rab protein bound to GDP. Sec4 in both states grossly resembles other G proteins bound to GDP and GppNHp. In Sec4-GppNHp, structural features common to active Rab proteins are observed. In Sec4-GDP, the switch I region is highly disordered and displaced relative to the switch I region of Ras-GDP. In two of the four molecules of Sec4-GDP in the asymmetric unit of the Sec4-GDP crystals, the switch II region adopts a conformation similar to that seen in the structure of the small G protein Ran bound to GDP. This allows residues threonine 76, glutamate 80, and arginine 81 of Sec4 to make contacts with other conserved residues and water molecules important for nucleotide binding. In the other two molecules in the asymmetric unit, these interactions do not take place. This structural variability in both the switch I and switch II regions of GDP-bound Sec4 provides a possible explanation for the high off-rate of GDP bound to Sec4, and suggests a mechanism for regulation of the GTPase cycle of Rab proteins by GDI proteins.