A common evolutionary origin of two elementary enzyme folds

The (beta alpha)(8)-barrel is the most frequent and most versatile fold among enzymes [H?cker et al., Curr. Opin. Biotechnol. 12 (2001) 376-381; Wierenga, FEBS Lett. 492 (2001) 193-198]. Structural and functional evidence suggests that (beta alpha)(8)-barrels evolved from an ancestral half-barrel, which consisted of four (beta alpha) units stabilized by dimerization [Lang et al., Science 289 (2000) 1546-550; H?cker et al., Nat. Struct. Biol. 8 (2001) 32-36; Gerlt and Babbitt, Nat. Struct. Biol. 8 (2001) 5-7]. Here, by performing a comprehensive database search, we detect a striking and unexpected structural and amino acid sequence similarity between (beta alpha)(4) half-barrels and members of the (beta alpha)(5) flavodoxin-like fold. These findings provoke the hypothesis that a large fraction of the modern-day enzymes evolved from a basic structural building block, which can be identified by a combination of sequence and structural analyses.     

The insect attractant 1-octen-3-ol is the natural ligand of bovine odorant-binding protein

Bovine odorant-binding protein (bOBP) is a dimeric lipocalin present in large amounts in the respiratory and olfactory nasal mucosa. The structure of bOBP refined at 2.0-A resolution revealed an elongated volume of electron density inside each buried cavity, indicating the presence of one (or several) naturally occurring copurified ligand(s) (Tegoni et al. (1996) Nat. Struct. Biol. 3, 863-867; Bianchet et al. (1996) Nat. Struct. Biol. 3, 934-939). In the present work, by combining mass spectrometry, x-ray crystallography (1.8-A resolution), and fluorescence, it has been unambiguously established that natural bOBP contains the racemic form of 1-octen-3-ol. This volatile substance is a typical component of bovine breath and in general of odorous body emanations of humans and animals. The compound 1-octen-3-ol is also an extremely potent olfactory attractant for many insect species, including some parasite vectors like Anopheles (Plasmodium) or Glossina (Trypanosoma). For the first time, a function can be assigned to an OBP, with a possible role of bOBP in the ecological relationships between bovine and insect species.     

Kinetics of tethering quaternary ammonium compounds to K(+) channels

Polymeric maleimido-quaternary ammonium (QA) compounds have been shown to function as molecular tape measures when covalently tethered to external cysteine residues of a Shaker K(+) channel (Blaustein R.O., P.A. Cole, C. Williams, and C. Miller. 2000. Nat. Struct. Biol. 7:309-311). For sufficiently long compounds, the cysteine-maleimide tethering reaction creates a high concentration, at the channel's pore, of a TEA-like moiety that irreversibly blocks current. This paper investigates a striking feature of the maleimide-cysteine tethering kinetics. Strong blockers-those that induce substantial levels (>80%) of irreversible inhibition of current-react with channel cysteines much more rapidly than weak blockers and, when delivered to channels with four cysteine targets, react with multiexponential kinetics. This behavior is shown to arise from the ability of a strong blocker to concentrate its maleimide end near a channel's cysteine target by exploiting the reversible pore-blocking affinity of its QA headgroup.     

Crystal structures of bovine odorant-binding protein in complex with odorant molecules.

The structure of bovine odorant-binding protein (bOBP) revealed a striking feature of a dimer formed by domain swapping [Tegoni, M., Ramoni, R., Bignetti, E., Spinelli, S. & Cambillau, C. (1996) Nat. Struct. Biol.3, 863-867; Bianchet, M.A., Bains, G., Pelosi, P., Pevsner, J., Snyder, S.H., Monaco, H.L. & Amzel, L.M. (1996) Nat. Struct. Biol.3, 934-939] and the presence of a naturally occuring ligand [Ramoni, R., Vincent, F., Grolli, S., Conti, V., Malosse, C., Boyer, F.D., Nagnan-Le Meillour, P., Spinelli, S., Cambillau, C. & Tegoni, M. (2001) J. Biol. Chem.276, 7150-7155]. These features led us to investigate the binding of odorant molecules with bOBP in solution and in the crystal. The behavior of odorant molecules in bOBP resembles that observed with porcine OBP (pOBP), although the latter is monomeric and devoid of ligand when purified. The odorant molecules presented K(d) values with bOBP in the micromolar range. Most of the X-ray structures revealed that odorant molecules interact with a common set of residues forming the cavity wall and do not exhibit specific interactions. Depending on the ligand and on the monomer (A or B), a single residue--Phe89--presents alternate conformations and might control cross-talking between the subunits. Crystal data on both pOBP and bOBP, in contrast with binding and spectroscopic studies on rat OBP in solution, reveal an absence of significant conformational changes involving protein loops or backbone. Thus, the role of OBP in signal triggering remains unresolved.     

Dll3 pudgy mutation differentially disrupts dynamic expression of somite genes

Mutations in the notch ligand delta-like 3 have been identified in both the pudgy mouse (Dll3(pu); Kusumi et al.: Nat Genet 19:274-278, 1998) and the human disorder spondylocostal dysostosis (SCD; Bulman et al.: Nat Genet 24:438-441, 2000), and a targeted mutation has been generated (Dll3(neo); Dunwoodie et al.: Development 129:1795-1806, 2002). Vertebral and rib malformations deriving from defects in somitic patterning are key features of these disorders. In the mouse, notch pathway genes such as Lfng, Hes1, Hes7, and Hey2 display dynamic patterns of expression in paraxial mesoderm, cycling in synchrony with somite formation (Aulehla and Johnson: Dev Biol 207:49-61, 1999; Forsberg et al.: Curr Biol 8:1027-1030, 1998; Jouve et al.: Development 127:1421-1429, 2000; McGrew et al.: Curr Biol 8:979-982, 1998; Nakagawa et al.: Dev Biol 216:72-84, 1999). We report here that the Dll3(pu) mutation has different effects on the expression of cycling (Lfng and Hes7) and stage-specific genes (Hey3 and Mesp2). This suggests a more complex situation than a single oscillatory mechanism in somitogenesis and provides an explanation for the unique radiological features of the human DLL3-type of SCD.     

Revisiting the catalytic CuZ cluster of nitrous oxide (N2O) reductase. Evidence of a bridging inorganic sulfur

Nitrous-oxide reductases (N2OR) catalyze the two-electron reduction of N(2)O to N(2). The crystal structure of N2ORs from Pseudomonas nautica (Pn) and Paracoccus denitrificans (Pd) were solved at resolutions of 2.4 and 1.6 A, respectively. The Pn N2OR structure revealed that the catalytic CuZ center belongs to a new type of metal cluster in which four copper ions are liganded by seven histidine residues. A bridging oxygen moiety and two other hydroxide ligands were proposed to complete the ligation scheme (Brown, K., Tegoni, M., Prudencio, M., Pereira, A. S., Besson, S., Moura, J. J. G., Moura, I., and Cambillau, C. (2000) Nat. Struct. Biol. 7, 191-195). However, in the CuZ cluster, inorganic sulfur chemical determination and the high resolution structure of Pd N2OR identified a bridging inorganic sulfur instead of an oxygen. This result reconciles the novel CuZ cluster with the hitherto puzzling spectroscopic data.     

Structural mechanisms of constitutive activation in the C5a receptors with mutations in the extracellular loops: molecular modeling study

Previously we demonstrated by random saturation mutagenesis a set of mutations in the extracellular (EC) loops that constitutively activate the C5a receptor (C5aR) (Klco et al., Nat Struct Mol Biol 2005;12:320-326; Klco et al., J Biol Chem 2006;281:12010-12019). In this study, molecular modeling revealed possible conformations for the extracellular loops of the C5a receptors with mutations in the EC2 loop or in the EC3 loop. Comparison of low-energy conformations of the EC loops defined two distinct clusters of conformations typical either for strongly constitutively active mutants of C5aR (the CAM cluster) or for nonconstitutively active mutants (the non-CAM cluster). In the CAM cluster, the EC3 loop was turned towards the transmembrane (TM) helical bundle and more closely interacted with EC2 than in the non-CAM cluster. This suggested a structural mechanism of constitutive activity where EC3 contacts EC2 leading to EC2 interactions with helix TM3, thus triggering movement of TM7 towards TM2 and TM3. The movement initiates rearrangement of the system of hydrogen bonds between TM2, TM3 and TM7 including formation of the hydrogen bond between the side chains of D82(2.50) in TM2 and N296(7.49) in TM7, which is crucial for formation of the activated states of the C5a receptors (Nikiforovich et al., Proteins: Struct Funct Gene 2011;79:787-802). Since the relative large length of EC3 in C5aR (13 residues) is comparable with those in many other members of rhodopsin family of GPCRs (13-19 residues), our findings might reflect general mechanisms of receptor constitutive activation. The very recent X-ray structure of the agonist-induced constitutively active mutant of rhodopsin (Standfuss et al., Nature 2011;471:656-660) is discussed in view of our modeling results.     

Analysis of the differences in the folding kinetics of structurally homologous proteins based on predictions of the gross features of residue contacts

It is a general notion that proteins with very similar three-dimensional structures would show very similar folding kinetics. However, recent studies reveal that the folding kinetic properties of some proteins contradict this thought (i.e., the members in a same protein family fold through different pathways). For example, it has been reported that some beta-proteins in the intracellular lipid-binding protein family fold through quite different pathways (Burns et al., Proteins 1998;33:107-118). Similar differences in folding kinetics are also observed in the members of the globin family (Nishimura et al., Nat Struct Biol 2000;7:679-686). In our study, we examine the possibility of predicting qualitative differences in folding kinetics of the intracellular lipid-binding proteins and two globin proteins (i.e., myoglobin and leghemoglobin). The problem is tackled by means of a contact map based on the average distance statistics between residues, the Average Distance Map (ADM), as constructed from sequence. The ADMs for the three proteins show overall similarity, but some local differences among maps are also observed. Our results demonstrate that some properties of the protein folding kinetics are consistent with local differences in the ADMs. We also discuss the general possibility of predicting folding kinetics from sequence information.     

Point mutants of c-raf-1 RBD with elevated binding to v-Ha-Ras

A mutational analysis of the Ras-binding domain (RBD) of c-Raf-1 identified three amino acid positions (Asn(64), Ala(85), and Val(88)) where amino acid substitution with basic residues increases the binding of RBD to recombinant v-Ha-Ras. The greatest increase in binding (6-9-fold) was observed with the A85K-RBD mutant. The elevated binding for the A85K-RBD and V88R-RBD mutants was also detected with Ras expressed in cultured mammalian cells, namely NIH-3T3 and BAF cells. None of the wild type residues in RBD positions Asn(64), Ala(85), and Val(88) have been previously implicated in the interaction with Ras (Block, C., Janknecht, R., Herrmann, C., Nassar, N., and Wittinghofer, A. (1996) Nat. Struct. Biol. 3, 244-251; Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1995) Nature 375, 554-560). The discovery of elevated binding among the mutants in these positions implies that additional RBD residues can be used to generate the Ras. RBD complex. These findings are of particular significance in the design of Ras antagonists based on the RBD prototype. The A85K-RBD mutant can be used to develop an assay for measuring the level of activated Ras in cultured cells; Sepharose-linked A85K-RBD.GST fusion protein served as an activation-specific probe to precipitate Ras.GTP but not Ras.GDP from epidermal growth factor-stimulated cells. A85K-RBD precipitates up to 5-fold more Ras.GTP from mammalian cells than wild type RBD.     

Genomic tagging of endogenous type IIbeta phosphatidylinositol 5-phosphate 4-kinase in DT40 cells reveals a nuclear localisation

Previous studies from acutely transfected HeLa cells have identified an acidic alpha-helix in the Type IIbeta PtdIns5P 4-kinase (PIPkin IIbeta) as a putative novel nuclear localisation sequence (Ciruela et al. Biochem. J. 364, 587-591 2000). However, some heterogeneity in cellular localisation was always observed, and other published aspects of PIPkin IIbeta physiology are more consistent with an extra-nuclear function. As a means of resolving whether the endogenous PIPkin IIbeta is nuclear, we have used the high homologous recombination frequency of DT40 cells to knock an epitope tag (Mosedale et al., Nat Struct Biol. 12, 763-771 2005) into one of the alleles of the DT40 PIPkin IIbeta gene. We show that PIPkin IIbeta is expressed as a tagged protein, is active as revealed by immunoprecipitation and enzyme assay, and that cellular fractionation reveals that it is indeed nuclear. Genomic tagging of endogenous proteins in DT40 cells is a technique that offers unique advantages in studying endogenous signalling proteins.     

Engineered intermonomeric disulfide bonds in the globular domain of Newcastle disease virus hemagglutinin-neuraminidase protein: implications for the mechanism of fusion promotion

The promotion of membrane fusion by Newcastle disease virus (NDV) requires an interaction between the viral hemagglutinin-neuraminidase (HN) and fusion (F) proteins, although the mechanism by which this interaction regulates fusion is not clear. The NDV HN protein exists as a tetramer composed of a pair of dimers. Based on X-ray crystallographic studies of the NDV HN globular domain (S. Crennell et al., Nat. Struct. Biol. 7:1068-1074, 2000), it was proposed that the protein undergoes a significant conformational change from an initial structure having minimal intermonomeric contacts to a structure with a much more extensive dimer interface. This conformational change was predicted to be integral to fusion promotion with the minimal interface form required to maintain F in its prefusion state until HN binds receptors. However, no evidence for such a conformational change exists for any other paramyxovirus attachment protein. To test the NDV model, we have engineered a pair of intermonomeric disulfide bonds across the dimer interface in the globular domain of an otherwise non-disulfide-linked NDV HN protein by the introduction of cysteine substitutions for residues T216 and D230. The disulfide-linked dimer is formed both intracellularly and in the absence of receptor binding and is efficiently expressed at the cell surface. The disulfide bonds preclude formation of the minimal interface form of the protein and yet enhance both receptor-binding activity at 37 degrees C and fusion promotion. These results confirm that neither the minimal interface form of HN nor the proposed drastic conformational change in the protein is required for fusion.     

Homoserine dehydrogenase from Saccharomyces cerevisiae: kinetic mechanism and stereochemistry of hydride transfer

Homoserine dehydrogenase (HSD), which is required for the synthesis of threonine, isoleucine and methionine in fungi, is a potential target for novel antifungal drugs. In order to design effective inhibitors, the kinetic mechanism of Saccharomyces cerevisiae HSD and the stereochemistry of hydride transfer were examined. Product inhibition experiments revealed that yeast HSD follows an ordered Bi Bi kinetic mechanism, where NAD(P)H must bind the enzyme prior to aspartate semialdehyde (ASA) and homoserine is released first followed by NAD(P)+. H-(1,2,4-triazol-3-yl)-D,L-alanine was an uncompetitive inhibitor of HSD with respect to NADPH (K(ii)=3.04+/-0.18 mM) and a noncompetitive inhibitor with respect to ASA (K(is)=1.64+/-0.36 mM, K(ii)=3.84+/-0.46 mM), in agreement with the proposed substrate order. Both kinetic isotope and viscosity experiments provided evidence for a very rapid catalytic step and suggest nicotinamide release to be primarily rate limiting. Incubation of HSD with stereospecifically deuterated NADP[2H] and subsaturating amounts of aspartate semialdehyde revealed that the pro-S NADPH hydride is transferred to the aldehyde. The pH dependence of steady state kinetic parameters indicate that ionizable groups with basic pKs may be involved in substrate binding, consistent with the observation of Lys223 at the enzyme active site in the recently determined 3D structure [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. These findings provide the requisite foundation for future exploitation of fungal HSD in inhibitor design.     

Lipids as targeting signals: lipid rafts and intracellular trafficking

Our view of biological membranes has evolved dramatically over the last few decades. In the bilayer model from Singer & Nicholson (Science 1972;175:720-731), both proteins and lipids freely diffuse within the plane of the membrane. Currently, however, membranes are viewed as a mosaic of different compartments or domains maintained by an active cytoskeleton network (Ritchie et al. Mol Membr Biol 2003; 20:13-18). Due to interactions between membrane components, several types of subdomains can form with different characteristics and functions. Lipids are likely to play an important role in the formation of so-called lipid-enriched microdomains or lipid rafts, adding another order of complexity to the membrane model. Rafts represent a type of domain wherein lipids of specific chemistry may dynamically associate with each other, to form platforms important for membrane protein sorting and construction of signaling complexes (Simons & Toomre. Nat Rev Mol Cell Biol 2000;1:31-39). Currently, there are several hypotheses concerning the nature of rafts (reviewed in (Edidin. Annu Rev Biophys Biomol Struct 2003;32: 257-283; Zurzolo et al. EMBO Rep 2003;4:1117-1121)). The most commonly cited one, proposed by Kai Simons (Simons & Ikonen. Nature 1997;387:569-572; Pralle et al. J Cell Biol 2000;148:997-1008), suggests that rafts are relatively small structures ( approximately 50 nm) enriched in cholesterol and sphingolipids within which associated proteins are likely to be concentrated. Another proposal (Anderson & Jacobson. Science 2002;296:1821-1825) suggests that rafts are constructed of lipid shells. These are small dynamic assemblies wherein 'raft' proteins are preferentially associated with certain types of lipids. These 'shells' are thermodynamically stable mobile entities in the plane of the membrane that are able to target the protein they encase to preexisting rafts/caveolae domains. In this review we summarize the data suggesting a specific role for lipid domains in intracellular trafficking and sorting and present a modification of the raft model that may help explain the observed phenomena.     

Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121

Bacterial fructosyltransferases (FTFs) are retaining-type glycosidases that belong to family 68 of glycoside hydrolases. Recently, the high-resolution 3D structure of the Bacillus subtilis levansucrase has been solved [Meng, G. and Futterer, K., Nat. Struct. Biol. 10 (2003) 935–941]. Based on this structure, the catalytic nucleophile, general acid/base catalyst, and transition state stabilizer were identified. However, a detailed characterization of site-directed mutants of the catalytic nucleophile has not been presented for any FTF enzyme. We have constructed site-directed mutants of the three putative catalytic residues of the Lactobacillus reuteri 121 levansucrase and inulosucrase and characterized the mutant proteins. Changing the putative catalytic nucleophiles D272 (inulosucrase) and D249 (levansucrase) into their amido counterparts resulted in a 1.5–4×10⁵ times reduction of total sucrase activity.     

A time-resolved iron-specific X-ray absorption experiment yields no evidence for an Fe2+ --> Fe3+ transition during QA- --> QB electron transfer in the photosynthetic reaction center

Previous time-resolved FTIR measurements suggested the involvement of an intermediary component in the electron transfer step Q(A)- --> Q(B) in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides [Remy and Gerwert (2003) Nat. Struct. Biol. 10, 637]. By a kinetic X-ray absorption experiment at the Fe K-edge we investigated whether oxidation occurs at the ferric non-heme iron located between the two quinones. In isolated reaction centers with a high content of functional Q(B), at a time resolution of 30 micros and at room temperature, no evidence for transient oxidation of Fe was obtained. However, small X-ray transients occurred, in a similar micro- to millisecond time range as in the IR experiments, which may point to changes in the Fe ligand environment due to the charges on Q(A)- and Q(B)-. In addition, VIS measurements agree with the IR data and do not exclude an intermediate in the Q(A)- --> Q(B) transition.     

The binding of phosphatidylcholine to the phosphatidylcholine transfer protein: affinity and role in folding

Bovine liver phosphatidylcholine transfer protein (PC-TP) has been expressed in Escherichia coli and purified to homogeneity from the cytosol fraction at a yield of 0.45 mg PC-TP per 10 mg total cytosolic protein. In addition, active PC-TP was obtained from inclusion bodies. An essential factor in the activation of PC-TP was phosphatidylcholine (PC) present in the folding buffer. PC-TP from the cytosol contains phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) with a preference for the di-monounsaturated species over the saturated species as determined by fast atom bombardment mass spectrometry (FAB-MS). By incubation with microsomal membranes the endogenous PE and PG were replaced by PC. Relative to the microsomal PC species composition, PC-TP bound preferentially C16:0/C20:4-PC and C16:0/C18:2-PC (twofold enriched) whereas the major microsomal species C18:0/C18:1-PC and C18:0/C18:2-PC were distinctly less bound. PC-TP is structurally homologous to the lipid-binding domain of the steroidogenic acute regulatory protein (Nat. Struct. Biol. 7 (2000) 408). Replacement of Lys(55) present in one of the beta-strands forming the lipid-binding site, with an isoleucine residue yielded an inactive protein. This suggests that Lys(55) be involved in the binding of the PC molecule.     

IA3, an aspartic proteinase inhibitor from Saccharomyces cerevisiae, is intrinsically unstructured in solution

IA(3) is a highly specific and potent 68-amino acid endogenous inhibitor of yeast proteinase A (YprA), and X-ray crystallographic studies have shown that IA(3) binds to YprA as an alpha-helix [Li, M., Phylip, L. H., Lees, W. E., Winther, J. R., Dunn, B. M., Wlodawer, A., Kay, J., and Gustchina, A. (2000) Nat. Struct. Biol. 7, 113-117]. Surprisingly, only residues 2-32 of IA(3) are seen in the X-ray structure, and the remaining residues are believed to be disordered in the complex. We have used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to show that IA(3) is unstructured in the absence of YprA. Specifically, IA(3) produced a CD spectrum characteristic of an unstructured peptide, and the (15)N HSQC NMR spectra of IA(3) were characteristic of a polypeptide lacking intrinsic structure. We characterized the unstructured state of IA(3) by using singular-value decomposition (SVD) to analyze the CD data in the presence of TFE, by fully assigning the unbound IA(3) protein by NMR and comparing the chemical shifts to published random-coil values, and by measuring (1)H-(15)N heteronuclear NOEs, which are all consistent with an unfolded protein. The IA(3) samples used for NMR analyses were active and inhibited YprA with an inhibition constant (K(i)) of 1.7 nM, and the addition of YprA led to a large spectral transition in IA(3). Calorimetric (ITC) data also show that the overall enthalpy of the interaction between IA(3) and YprA is exothermic.