Aspzincin, a family of metalloendopeptidases with a new zinc-binding motif. Identification of new zinc-binding sites (His(128), His(132), and Asp(164)) and three catalytically crucial residues (Glu(129), Asp(143), and Tyr(106)) of deuterolysin from Aspergillus oryzae by site-directed mutagenesis.

Deuterolysin (EC 3.4.24.39; formerly designated as neutral proteinase II) from Aspergillus oryzae, which contains 1 g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. Active-site determination of the recombinant enzyme expressed in Escherichia coli was performed by site-directed mutagenesis. Substitutions of His(128) and His(132) with Arg, of Glu(129) with Gln or Asp, of Asp(143) with Asn or Glu, of Asp(164) with Asn, and of Tyr(106) with Phe resulted in almost complete loss of the activity of the mutant enzymes. It can be concluded that His(128), His(132), and Asp(164) provide the Zn(2+) ligands of the enzyme according to a (65)Zn binding assay. Based on site-directed mutagenesis experiments, it was demonstrated that the three essential amino acid residues Glu(129), Asp(143), and Tyr(106) are catalytically crucial residues in the enzyme. Glu(129) may be implicated in a central role in the catalytic function. We conclude that deuterolysin is a member of a family of Zn(2+) metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand.     

Identification of the zinc binding ligands and the catalytic residue in human aspartoacylase, an enzyme involved in Canavan disease

Canavan disease is an autosomal-recessive neurodegenerative disorder caused by a lack of aspartoacylase, the enzyme that degrades N-acetylaspartate (NAA) into acetate and aspartate. With a view to studying the mechanisms underlying the action of human aspartoacylase (hASP), this enzyme was expressed in a heterologous Escherichia coli system and characterized. The recombinant protein was found to have a molecular weight of 36 kDa and kinetic constants K(m) and k(cat) of 0.20 +/- 0.03 mM and 14.22 +/- 0.48 s(-1), respectively. Sequence alignment showed that this enzyme belongs to the carboxypeptidase metalloprotein family having the conserved motif H(21)xxE(24)(91aa)H(116). We further investigated the active site of hASP by performing modelling studies and site-directed mutagenesis. His21, Glu24 and His116 were identified here for the first time as the residues involved in the zinc-binding process. In addition, mutations involving the Glu178Gln and Glu178Asp residues resulted in the loss of enzyme activity. The finding that wild-type and Glu178Asp have the same K(m) but different k(cat) values confirms the idea that the carboxylate group contributes importantly to the enzymatic activity of aspartoacylase.     

The Study of the Metal-Binding Region in Human Growth Hormone Using Immobilized Metal Ion Affinity Gel-Electrophoresis

The zinc(II)-binding affinities of recombinant human growth hormone and two its mutants, 14–33 and 14–95, were studied using Immobilized Metal Ion Affinity Gel-electrophoresis (IMAG). The mutant hormones, composed of polypeptide chain segments of the human and porcine growth hormones, lacked His18, which may be crucial for binding of the intact hormone to the transition metal ions. The mutations did not affect the affinity of human growth hormone to immobilized zinc ions; the structural analysis implied that the human growth hormone contains two IDA–Zn(II) potential sorption sites formed by amino acid residues His21, Asp171, and Glu174 and/or His18 and Glu174.     

Crystal structure of MqnD (TTHA1568), a menaquinone biosynthetic enzyme from Thermus thermophilus HB8

In many microorganisms, menaquinone is an essential lipid-soluble electron carrier. Recently, an alternative menaquinone biosynthetic pathway was found in some microorganisms [Hiratsuka, T., Furihata, K., Ishikawa, J., Yamashita, H., Itoh, N., Seto, H., Dairi, T., 2008. An alternative menaquinone biosynthetic pathway operating in microorganisms. Science 321, 1670–1673]. Here, we report the 1.55 Å crystal structure of MqnD (TTHA1568) from Thermus thermophilus HB8, an enzyme within the alternative menaquinone biosynthetic pathway. The structure comprises two domains with α/β structures, a large domain and a small domain. L(+)-Tartaric acid was bound to the pocket between the two domains, suggesting that this pocket is a putative active site. The conserved glycine residues at positions 78, 80 and 82 seem to act as hinges, allowing the substrate to access the pocket. Highly conserved residues, such as Asp14, Asp38, Asn43, Ser57, Thr107, Ile144, His145, Glu146, Leu176 and Tyr234, are located at this pocket, suggesting that these residues are involved in substrate binding and/or catalysis, and especially, His145 could function as a catalytic base. Since humans and their commensal intestinal bacteria, including lactobacilli, lack the alternative menaquinone biosynthetic pathway, this enzyme in pathogenic species, such as Helicobacter pylori and Campylobacter jejuni, is an attractive target for the development of chemotherapeutics. This high-resolution structure may contribute toward the development of its inhibitors.     

Evidence for Restricted Reactivity of ADAMDEC1 with Protein Substrates and Endogenous Inhibitors

ADAMDEC1 is a proteolytically active metzincin metalloprotease displaying rare active site architecture with a zinc-binding Asp residue (Asp-362). We previously demonstrated that substitution of Asp-362 for a His residue, thereby reconstituting the canonical metzincin zinc-binding environment with three His zinc ligands, increases the proteolytic activity. The protease also has an atypically short domain structure with an odd number of Cys residues in the metalloprotease domain. Here, we investigated how these rare structural features in the ADAMDEC1 metalloprotease domain impact the proteolytic activity, the substrate specificity, and the effect of inhibitors. We identified carboxymethylated transferrin (Cm-Tf) as a new ADAMDEC1 substrate and determined the primary and secondary cleavage sites, which suggests a strong preference for Leu in the P1′ position. Cys³⁹², present in humans but only partially conserved within sequenced ADAMDEC1 orthologs, was found to be unpaired, and substitution of Cys³⁹² for a Ser increased the reactivity with α₂-macroglobulin but not with casein or Cm-Tf. Substitution of Asp³⁶² for His resulted in a general increase in proteolytic activity and a change in substrate specificity was observed with Cm-Tf. ADAMDEC1 was inhibited by the small molecule inhibitor batimastat but not by tissue inhibitor of metalloproteases (TIMP)-1, TIMP-2, or the N-terminal inhibitory domain of TIMP-3 (N-TIMP-3). However, N-TIMP-3 displayed profound inhibitory activity against the D362H variants with a reconstituted consensus metzincin zinc-binding environment. We hypothesize that these unique features of ADAMDEC1 may have evolved to escape from inhibition by endogenous metalloprotease inhibitors.     

Role of nucleotidyltransferase motifs I, III and IV in the catalysis of phosphodiester bond formation by Chlorella virus DNA ligase

ATP-dependent DNA ligases catalyze the sealing of 5′-phosphate and 3′-hydroxyl termini at DNA nicks by means of a series of three nucleotidyl transfer steps. Here we have analyzed by site-directed mutagenesis the roles of conserved amino acids of Chlorella virus DNA ligase during the third step of the ligation pathway, which entails reaction of the 3′-OH of the nick with the DNA–adenylate intermediate to form a phosphodiester and release AMP. We found that Asp65 and Glu67 in nucleotidyltransferase motif III and Glu161 in motif IV enhance the rate of step 3 phosphodiester formation by factors of 20, 1000 and 60, respectively. Asp29 and Arg32 in nucleotidyltransferase motif I enhance the rate of step 3 by 60-fold. Gel shift analysis showed that mutations of Arg32 and Asp65 suppressed ligase binding to a pre-adenylated nick, whereas Asp29, Glu67 and Glu161 mutants bound stably to DNA–adenylate. We infer that Asp29, Glu67 and Glu161 are involved directly in the step 3 reaction. In several cases, the effects of alanine or conservative mutations on step 3 were modest compared to their effects on the composite ligation reaction and individual upstream steps. These results, in concert with available crystallographic data, suggest that the active site of DNA ligase is remodeled during the three steps of the pathway and that some of the catalytic side chains play distinct roles at different stages.     

N-Acetylglucosaminidases from CAZy Family GH3 Are Really Glycoside Phosphorylases,Thereby Explaining Their Use of Histidine as an Acid/Base Catalyst in Place of Glutamic Acid

CAZy glycoside hydrolase family GH3 consists primarily of stereochemistry-retaining β-glucosidases but also contains a subfamily of β-N-acetylglucosaminidases. Enzymes from this subfamily were recently shown to use a histidine residue within a His-Asp dyad contained in a signature sequence as their catalytic acid/base residue. Reasons for their use of His rather than the Glu or Asp found in other glycosidases were not apparent. Through studies on a representative member, the Nag3 β-N-acetylglucosaminidase from Cellulomonas fimi, we now show that these enzymes act preferentially as glycoside phosphorylases. Their need to accommodate an anionic nucleophile within the enzyme active site explains why histidine is used as an acid/base catalyst in place of the anionic glutamate seen in other GH3 family members. Kinetic and mechanistic studies reveal that these enzymes also employ a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate, which was directly detected by mass spectrometry. Phosphate has no effect on the rates of formation of the glycosyl-enzyme intermediate, but it accelerates turnover of the N-acetylglucosaminyl-enzyme intermediate ∼3-fold, while accelerating turnover of the glucosyl-enzyme intermediate several hundredfold. These represent the first reported examples of retaining β-glycoside phosphorylases, and the first instance of free β-GlcNAc-1-phosphate in a biological context.     

Mitochondrial phosphate transport protein. Reversions of inhibitory conservative mutations identify four helices and a nonhelix protein segment with transmembrane interactions and Asp39, Glu137, and Ser158 as nonessential for transport

The mitochondrial phosphate transport protein (PTP) has six (A--F) transmembrane (TM) helices per subunit of functional homodimer with all mutations referring to the subunit of the homodimer. In earlier studies, conservative replacements of several residues located either at the matrix end (Asp39/helix A, Glu137/helix C, Asp236/helix E) or at the membrane center (His32/helix A, Glu136/helix C) of TM helices yielded inactive single mutation PTPs. Some of these residues were suggested to act as phosphate ligands or as part of the proton cotransport path. We now show that the mutation Ser158Thr, not part of a TM helix but located near the center of the matrix loop (Ile141--Ser171) between TM helices C and D, inactivates PTP and is thus also functionally relevant. On the other side of the membrane, the single mutation Glu192Asp at the intermembrane space end of TM helix D yields a PTP with 33% wild-type activity. We constructed double mutants by adding this mutation to the six transport-inactivating mutations. Transport was detected only in those with Asp39Asn, Glu137Gln, or Ser158Thr. We conclude that TM helix D can interact with TM helices A and C and matrix loop Ile141--Ser171 and that Asp39, Glu137, and Ser158 are not essential for phosphate transport. Since our results are consistent with residues present in all 12 functionally identified members of the mitochondrial transport protein (MTP) family, they lead to a general rule that specifies MTP residue types at 7 separate locations. The conformations of all the double mutation PTPs (except that with the matrix loop Ser158Thr) are significantly different from those of the single mutation PTPs, as indicated by their very low liposome incorporation efficiency and their requirement for less detergent (Triton X-100) to stay in solution. These dramatic conformational differences also suggest an interaction between TM helices D and E. The results are discussed in terms of TM helix movements and changes in the PTP monomer/dimer ratio.     

Human nonsense-mediated mRNA decay factor UPF2 interacts directly with eRF3 and the SURF complex

Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that regulates gene expression and mRNA quality. A complex network of macromolecular interactions regulates NMD initiation, which is only partially understood. According to prevailing models, NMD begins by the assembly of the SURF (SMG1–UPF1–eRF1–eRF3) complex at the ribosome, followed by UPF1 activation by additional factors such as UPF2 and UPF3. Elucidating the interactions between NMD factors is essential to comprehend NMD, and here we demonstrate biochemically and structurally the interaction between human UPF2 and eukaryotic release factor 3 (eRF3). In addition, we find that UPF2 associates with SURF and ribosomes in cells, in an UPF3-independent manner. Binding assays using a collection of UPF2 truncated variants reveal that eRF3 binds to the C-terminal part of UPF2. This region of UPF2 is partially coincident with the UPF3-binding site as revealed by electron microscopy of the UPF2–eRF3 complex. Accordingly, we find that the interaction of UPF2 with UPF3b interferes with the assembly of the UPF2–eRF3 complex, and that UPF2 binds UPF3b more strongly than eRF3. Together, our results highlight the role of UPF2 as a platform for the transient interactions of several NMD factors, including several components of SURF.     

Role of two active site Glu residues in the molecular action of botulinum neurotoxin endopeptidase

Botulinum neurotoxin type A (BoNT/A) light chain (LC) is a zinc endopeptidase that causes neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. The X-ray crystal structure of the toxin reveals that His223 and His227 of the Zn(2+) binding motif HEXXH directly coordinate the active site zinc. Two Glu residues (Glu224 and Glu262) are also part of the active site, with Glu224 coordinating the zinc via a water molecule whereas Glu262 coordinates the zinc directly as the fourth ligand. In the past we have investigated the topographical role of Glu224 by replacing it with Asp thus reducing the side chain length by 1.4 A that reduced the endopeptidase activity dramatically [L. Li, T. Binz, H. Niemann, and B.R. Singh, Probing the role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain, Biochemistry 39 (2000) 2399-2405]. In this study we have moved the Glu 224 laterally by a residue (HXEXH) to assess its positional influence on the endopeptidase activity, which was completely lost. The functional implication of Glu262 was investigated by replacing this residue with aspartate and glutamine using site-directed mutagenesis. Substitution of Glu262 with Asp resulted in a 3-fold decrease in catalytic efficiency. This mutation did not induce any significant structural alterations in the active site and did not interfere with substrate binding. Substitution of Glu262 with Gln however, dramatically impaired the enzymatic activity and this is accompanied by global alterations in the active site conformation in terms of topography of aromatic amino acid residues, zinc binding, and substrate binding, resulting from the weakened interaction between the active site zinc and Gln. These results suggest a pivotal role of the negatively charged carboxyl group of Glu262 which may play a critical role in enhancing the stability of the active site with strong interaction with zinc. The zinc may thus play structural role in addition to its catalytic role.     

Mutational analysis of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) nucleotide pyrophosphatase/phosphodiesterase shows the role of six amino acids in the catalytic mechanism

Nucleotide pyrophosphatases/phosphodiesterases (NPPs, PF01663) release nucleoside 5′-monophosphates from a wide range of nucleotide substrates. Only very recently, the first plant members of the NPP family were characterised (Joye et al. J Cereal Sci 51: 326–336, 2010), and little is known about their substrate-specifying residues. We elucidated the role of six amino acid residues of the recently identified and characterised Triticum aestivum L. NPP (Joye et al. J Cereal Sci 51: 326–336, 2010). Substitution of the highly conserved catalytic Thr132 into Ser or Ala completely abolished enzyme activity. Mutation of a highly conserved His255 residue into an apolar Ala suprisingly increased enzyme activity against most phosphodiester substrates. Four other residues moderately to highly conserved over NPPs of different organisms were studied as well. Mutation of the Asn153, Asn165 and Glu199 into an Arg, Ser and Asp residue, respectively, increased the relative enzyme activity against p-nitrophenyl phosphate. Furthermore, mutation of Phe194 into Ser increased the relative enzyme activity against adenosine 5′-monophosphate-containing substrates, although the overall enzyme activity of this mutant enzyme decreased. We conclude that the structural requirements and the conservation of the amino acids of the catalytic site of TaNPPr and, by extension, probably of all NPPs, are very stringent.     

NAR Breakthrough Article: Structure of human RNA N6-methyladenine demethylase ALKBH5 provides insights into its mechanisms of nucleic acid recognition and demethylation

ALKBH5 is a 2-oxoglutarate (2OG) and ferrous iron-dependent nucleic acid oxygenase (NAOX) that catalyzes the demethylation of N⁶-methyladenine in RNA. ALKBH5 is upregulated under hypoxia and plays a role in spermatogenesis. We describe a crystal structure of human ALKBH5 (residues 66–292) to 2.0 Å resolution. ALKBH5_66–292 has a double-stranded β-helix core fold as observed in other 2OG and iron-dependent oxygenase family members. The active site metal is octahedrally coordinated by an HXD…H motif (comprising residues His204, Asp206 and His266) and three water molecules. ALKBH5 shares a nucleotide recognition lid and conserved active site residues with other NAOXs. A large loop (βIV–V) in ALKBH5 occupies a similar region as the L1 loop of the fat mass and obesity-associated protein that is proposed to confer single-stranded RNA selectivity. Unexpectedly, a small molecule inhibitor, IOX3, was observed covalently attached to the side chain of Cys200 located outside of the active site. Modelling substrate into the active site based on other NAOX–nucleic acid complexes reveals conserved residues important for recognition and demethylation mechanisms. The structural insights will aid in the development of inhibitors selective for NAOXs, for use as functional probes and for therapeutic benefit.     

Importance of the conserved residues in the peptidoglycan glycosyltransferase module of the class A penicillin-binding protein 1b of Escherichia coli

The peptidoglycan glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs catalyze glycan chain elongation of the bacterial cell wall. These enzymes belong to the GT51 family, are characterized by five conserved motifs, and have some fold similarity with the phage lambda lysozyme. In this work, we have systematically modified all the conserved amino acid residues of the GT module of Escherichia coli class A PBP1b by site-directed mutagenesis and determined their importance for the in vivo and in vitro activity and the thermostability of the protein. To get an insight into the GT active site of this paradigm enzyme, a model of PBP1b GT domain was constructed based on the available crystal structures (PDB codes 2OLV and 2OLU). The data show that in addition to the essential glutamate residues Glu233 of motif 1 and Glu290 of motif 3, the residues Phe237 and His240 of motif 1 and Gly264, Thr267, Gln271, and Lys274 of motif 2, all located in the catalytic cavity of the GT domain, are essential for the in vitro enzymatic activity of the PBP1b and for its in vivo functioning. Thus, the first three conserved motifs contain most of the residues that are required for the GT activity of the PBP1b. The residues Asp234, Phe237, His240, Thr267, and Gln271 are proposed to maintain the structure of the active site and the positioning of the catalytic Glu233.     

UPF201 Archaeal Specific Family Members Reveal Structural Similarity to RNA-Binding Proteins but Low Likelihood for RNA-Binding Function

We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54) to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10–40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel β-sheet and five α-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation.     

Re-evaluation of amino acid sequence and structural consensus rules for cysteine-nitric oxide reactivity

Nitric oxide (NO), produced in different cell types through the conversion of L-arginine into L-citrulline by the enzyme NO synthase, has been proposed to exert its action in several physiological and pathological events. The great propensity for nitrosothiol formation and breakdown represents a mechanism which modulates the action of macromolecules containing NO-reactive Cys residues at their active centre and/or allosteric sites. Based on the human haemoglobin (Hb) structure and accounting for the known acid-base catalysed Cys beta93-nitrosylation and Cys beta393NO-denitrosylation processes, the putative amino acid sequence (Lys/Arg/His/Asp/Glu)Cys(Asp/Glu) (sites -1, 0, and + 1, respectively) has been proposed as the minimum consensus motif for Cys-NO reactivity. Although not found in human Hb, the presence of a polar amino acid residue (Gly/Ser/Thr/Cys/Tyr/Asn/Gln) at the -2 position has been observed in some NO-reactive protein sequences (e.g., NMDA receptors). However, the most important component of the tri- or tetra-peptide consensus motif has been recognised as the Cys(Asp/Glu) pair [Stamler et al., Neuron (1997) 18, 691-696]. Here, we analyse the three-dimensional structure of several proteins containing NO-reactive Cys residues, and show that their nitrosylation and denitrosylation processes may depend on the Cys-Sy atomic structural microenvironment rather than on the tri- or tetra-peptide sequence consensus motif.     

Purification and properties of a β-glycosidase purified from midgut cells of Spodoptera frugiperda (Lepidoptera) larvae

Two β-glycosidases (BG) (Mr 47,000 and Mr 50,000) were purified from Spodoptera frugiperda (Lepidoptera: Noctuidae) midguts. These two polypeptides associate or dissociate depending on the medium ionic strength. The Mr 47,000 BG probably has two active sites. One of the putative active sites (cellobiase site) hydrolyses p-nitrophenyl β- -glucoside (NPβGlu) (79% of the total activity in saturated enzyme), cellobiose, amygdalin and probably also cellotriose, cellotetraose and cellopentaose. The cellobiase site has four subsites for glucose residue binding, as can be deduced from cellodextrin cleavage data. The enzymatic activity in this site is abolished after carbodiimide modification at pH 6.0. Since the inactivation is reduced in the presence of cellobiose, the results suggest the presence of a carboxylate as a catalytic group. The other active site of Mr 47,000 BG (galactosidase site) hydrolyses p-nitrophenyl β- -galactoside (NPβGal) better than NPβGlu, cleaves glucosylceramide and lactose and is unable to act on cellobiose, cellodextrins and amygdalin. This active site is not modified by carbodiimide at pH 6.0.The Mr 47,000 BG N-terminal sequence has high identity to plant β-glycosidases and to mammalian lactase–phlorizin hydrolase, and contains the QIEGA motif, characteristic of the family of glycosyl hydrolases. The putative physiological role of this enzyme is the digestion of glycolipids (galactosidase site) and di- and oligosaccharides (cellobiase site) derived from hemicelluloses, thus resembling mammalian lactase–phlorizin hydrolase.     

Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change.     

A model for how Gβγ couples Gα to GPCR

Representing ∼5% of the human genome, G-protein-coupled receptors (GPCRs) are a primary target for drug discovery; however, the molecular details of how they couple to heterotrimeric G protein subunits are incompletely understood. Here, I propose a hypothetical initial docking model for the encounter between GPCR and Gβγ that is defined by transient interactions between the cytosolic surface of the GPCR and the prenyl moiety and the tripeptide motif, asparagine–proline–phenylalanine (NPF), in the C-terminus of the Gγ subunit. Analysis of class A GPCRs reveals a conserved NPF binding site formed by the interaction of the TM1 and H8. Functional studies using differentially prenylated proteins and peptides further suggest that the intracellular hydrophobic core of the GPCR is a prenyl binding site. Upon binding TM1 and H8 of GPCRs, the propensity of the C-terminal region of Gγ to convert into an α helix allows it to extend into the hydrophobic core of the GPCR, facilitating the GPCR active state. Conservation of the NPF motif in Gγ isoforms and interacting residues in TM1 and H8 suggest that this is a general mechanism of GPCR–G protein signaling. Analysis of the rhodopsin dimer also suggests that Gγ–rhodopsin interactions may facilitate GPCR dimer transactivation.     

Crystal structure of the ββα-Me type II restriction endonuclease Hpy99I with target DNA

The ββα-Me restriction endonuclease (REase) Hpy99I recognizes the CGWCG target sequence and cleaves it with unusual stagger (five nucleotide 5′-recessed ends). Here we present the crystal structure of the specific complex of the dimeric enzyme with DNA. The Hpy99I protomer consists of an antiparallel β-barrel and two β4α2 repeats. Each repeat coordinates a structural zinc ion with four cysteine thiolates in two CXXC motifs. The ββα-Me region of the second β4α2 repeat holds the catalytic metal ion (or its sodium surrogate) via Asp148 and Asn165 and activates a water molecule with the general base His149. In the specific complex, Hpy99I forms a ring-like structure around the DNA that contacts DNA bases on the major and minor groove sides via the first and second β4α2 repeats, respectively. Hpy99I interacts with the central base pair of the recognition sequence only on the minor groove side, where A:T resembles T:A and G:C is similar to C:G. The Hpy99I–DNA co-crystal structure provides the first detailed illustration of the ββα-Me site in REases and complements structural information on the use of this active site motif in other groups of endonucleases such as homing endonucleases (e.g. I-PpoI) and Holliday junction resolvases (e.g. T4 endonuclease VII).