A general method for greatly improving the affinity of antibodies by using combinatorial libraries.

Look-through mutagenesis (LTM) is a multidimensional mutagenesis method that simultaneously assesses and optimizes combinatorial mutations of selected amino acids. The process focuses on a precise distribution within one or more complementarity determining region (CDR) domains and explores the synergistic contribution of amino acid side-chain chemistry. LTM was applied to an anti-TNF-alpha antibody, D2E7, which is a challenging test case, because D2E7 was highly optimized (K(d) = 1 nM) by others. We selected and incorporated nine amino acids, representative of the major chemical functionalities, individually at every position in each CDR and across all six CDRs (57 aa). Synthetic oligonucleotides, each introducing one amino acid mutation throughout the six CDRs, were pooled to generate segregated libraries containing single mutations in one, two, and/or three CDRs for each V(H) and V(L) domain. Corresponding antibody libraries were displayed on the cell surface of yeast. After positive binding selection, 38 substitutions in 21 CDR positions were identified that resulted in higher affinity binding to TNF-alpha. These beneficial mutations in both V(H) and V(L) were represented in two combinatorial beneficial mutagenesis libraries and selected by FACS to produce a convergence of variants that exhibit between 500- and 870-fold higher affinities. Importantly, these enhanced affinities translate to a 15- to 30-fold improvement in in vitro TNF-alpha neutralization in an L929 bioassay. Thus, this LTM/combinatorial beneficial mutagenesis strategy generates a comprehensive energetic map of the antibody-binding site in a facile and rapid manner and should be broadly applicable to the affinity maturation of antibodies and other proteins.

Heavy metal transport by AtHMA4 involves the N-terminal degenerated metal binding domain and the C-terminal His11 stretch.

The Arabidopsis thaliana AtHMA4 is a P1B-type ATPase that clusters with the Zn/Cd/Pb/Co subgroup. It has been previously shown, by heterologous expression and the study of AtHMA4 knockout or overexpressing lines in Arabidopsis , that AtHMA4 is implicated in zinc homeostasis and cadmium tolerance. Here, we report the study of the heterologous expression of AtHMA4 in the yeast Saccharomyces cerevisiae. AtHMA4 expression resulted in an increased tolerance to Zn, Cd and Pb and to a phenotypic complementation of hypersensitive mutants. In contrast, an increased sensitivity towards Co was observed. An AtHMA4::GFP fusion protein was observed in endocytic vesicles and at the yeast plasma membrane. Mutagenesis of the cysteine and glutamate residues from the N-ter degenerated heavy metal binding domain impaired the function of AtHMA4. It was also the case when the C-ter His11 stretch was deleted, giving evidence that these amino acids are essential for the AtHMA4 binding/translocation of metals.

The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography.

CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2'-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme-NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274-->Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R-N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 degrees C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 A resolution.

Use of photoaffinity labeling and site-directed mutagenesis for identification of the key residue responsible for extraordinarily high affinity binding of UCN-01 in human alpha1-acid glycoprotein.

7-Hydroxystaurosporine (UCN-01) is a protein kinase inhibitor anticancer drug currently undergoing a phase II clinical trial. The low distribution volumes and systemic clearance of UCN-01 in human patients have been found to be caused in part by its extraordinarily high affinity binding to human alpha1-acid glycoprotein (hAGP). In the present study, we photolabeled hAGP with [3H]UCN-01 without further chemical modification. The photolabeling specificity of [3H]UCN-01 was confirmed by findings in which other hAGP binding ligands inhibited formation of covalent bonds between hAGP and [3H]UCN-01. The amino acid sequence of the photolabeled peptide was concluded to be SDVVYTDXK, corresponding to residues Ser-153 to Lys-161 of hAGP. No PTH derivatives were detected at the 8th cycle, which corresponded to the 160th Trp residue. This strongly implies that Trp-160 was photolabeled by [3H]UCN-01. Three recombinant hAGP mutants (W25A, W122A, and W160A) and wild-type recombinant hAGP were photolabeled by [3H]UCN-01. Only mutant W160A showed a marked decrease in the extent of photoincorporation. These results strongly suggest that Trp-160 plays a prominent role in the high affinity binding of [3H]UCN-01 to hAGP. A docking model of UCN-01 and hAGP around Trp-160 provided further details of the binding site topology.

Transcriptional regulation of the human DNA methyltransferase 3A and 3B genes by Sp3 and Sp1 zinc finger proteins.

The DNMT3A (DNA methyltransferase 3A) and DNMT3B genes encode putative de novo methyltransferases and show complex transcriptional regulation in the presence of three and two different promoters respectively. All promoters of DNMT3A and DNMT3B lack typical TATA sequences adjacent to their transcription start sites and contain several Sp1-binding sites. The importance of these Sp1-binding sites was demonstrated by using a GC-rich DNA-binding protein inhibitor, mithramycin A, i.e. on the basis of decrease in the promoter activities and mRNA expression levels of DNMT3A and DNMT3B. Overexpression of Sp1 and Sp3 up-regulated the promoter activities of these two genes. The physical binding of Sp1 and Sp3 to DNMT3A and DNMT3B promoters was confirmed by a gel shift assay. Interestingly, Sp3 overexpression in HEK-293T cells (human embryonic kidney 293T cells) resulted in 3.3- and 4.0-fold increase in DNMT3A and DNMT3B mRNA expression levels respectively by quantitative reverse transcriptase-PCR, whereas Sp1 overexpression did not. Furthermore, an antisense oligonucleotide to Sp3 significantly decreased the mRNA levels of DNMT3A and DNMT3B. These results indicate the functional importance of Sp proteins, particularly Sp3, in the regulation of DNMT3A and DNMT3B gene expression.

Human xylosyltransferase I: functional and biochemical characterization of cysteine residues required for enzymic activity.

XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure-function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561-->Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.

Novel interaction partners of the TPR/MET tyrosine kinase.

A large variety of biological processes is mediated by stimulation of the receptor tyrosine kinase MET. Screening a mouse embryo cDNA library, we were able to identify several novel, putative intracellular TPR/MET-substrates: SNAPIN, DCOHM, VAV-1, Sorting nexin 2, Death associated protein kinase 3, SMC-1, Centromeric protein C, and hTID-1. Interactions as identified by yeast two-hybrid analysis were validated in vitro and in vivo by mammalian two-hybrid studies, a far-western assay and coimmunoprecipitation. Participation in apoptosis-regulating mechanisms through interaction with DAPK-3 and cell cycle control via binding to nuclear proteins such as CENPC and SMC-1 are possible new aspects of intracellular MET signaling.

Nucleotide specificity of HIV-1 reverse transcriptases with amino acid substitutions affecting Ala-114.

Ala-114, together with Asp-113, Tyr-115 and Gln-151, form the pocket that accommodates the 3'-OH of the incoming dNTP in the HIV-1 RT (reverse transcriptase). Four mutant RTs having serine, glycine, threonine or valine instead of Ala-114 were obtained by site-directed mutagenesis. While mutants A114S and A114G retained significant DNA polymerase activity, A114T and A114V showed very low catalytic efficiency in nucleotide incorporation assays, due to their high apparent K(m) values for dNTP. Discrimination between AZTTP (3'-azido-3'-deoxythymidine triphosphate) and dTTP was not significantly affected by mutations A114S and A114G in assays carried out with heteropolymeric template/primers. However, both mutants showed decreased susceptibility to AZTTP when poly(rA)/(dT)16 was used as substrate. Steady-state kinetic analysis of the incorporation of ddNTPs compared with dNTPs showed that substituting glycine for Ala-114 produced a 5-6-fold increase in the RT's ability to discriminate against ddNTPs (including the physiologically relevant metabolites of zalcitabine and didanosine), a result that was confirmed in primer-extension assays. In contrast, A114S and A114V showed wild-type ddNTP/dNTP discrimination efficiencies. Discrimination against ribonucleotides was not affected by mutations at position 114. Misinsertion and mispair extension fidelity assays as well as determinations of G-->A mutation frequencies using a lacZ complementation assay showed that, unlike Tyr-115 or Gln-151 mutants, the fidelity of HIV-1 RT was not largely affected by substitutions of Ala-114. The role of the side-chain of Ala-114 in ddNTP/dNTP discrimination appears to be determined by its participation in van der Waals interactions with the ribose moiety of the incoming nucleotide.

Transcriptional regulation of thioredoxin reductase 1 expression by cadmium in vascular endothelial cells: role of NF-E2-related factor-2.

Thioredoxin reductase (TrxR) is a selenoprotein that catalyzes the reduction of the active site disulfide of thioredoxin (Trx), which regulates the redox status of the cells. In the present study, we found that TrxR1, one of the three TrxR isozymes, was induced by cadmium as well as tumor necrosis factor alpha (TNFalpha) in bovine arterial endothelial cells (BAEC), and investigated the mechanism of cadmium-induced TrxR1 expression. We here showed that cadmium, differently from TNFalpha, enhanced the promoter activity of the 5'-flanking region of human TrxR1 gene (nucleotides -1692 to +49). Deletion and site-directed mutation of antioxidant responsive element (ARE) (nucleotides -62 to -48) in this region abolished the response to cadmium. Overexpression of NF-E2-related factor-2 (Nrf2) augmented the TrxR1 promoter activity. In contrast, overexpression of the dominant negative mutant of Nrf2 suppressed cadmium-induced activation of TrxR1 promoter through the ARE. Chromatin immunoprecipitation (ChIP) assays showed that anti-Nrf2 antibody precipitated ARE from the chromatin of the cadmium-treated cells. These results indicated that cadmium-induced TrxR1 gene expression is mediated by the activation of Nrf2 transcription factor and its binding to ARE in the TrxR1 gene promoter. We further found that in addition to cadmium, the activators of Nrf2, such as diethyl maleate (DEM) and arsenite, induced both TrxR1 and Trx gene expression in BAEC. Nrf2 might play an important role in the regulation of the cellular Trx system consisting of Trx and TrxR.

Uterus hyperplasia and increased carcinogen-induced tumorigenesis in mice carrying a targeted mutation of the Chk2 phosphorylation site in Brca1.

The tumor suppressor BRCA1 contains multiple functional domains that interact with many proteins. After DNA damage, BRCA1 is phosphorylated by CHK2 at serine 988, followed by a change in its intracellular location. To study the functions of CHK2-dependent phosphorylation of BRCA1, we generated a mouse model carrying the mutation S971A (S971 in mouse Brca1 corresponds to S988 in human BRCA1) by gene targeting. Brca1(S971A/S971A) mice were born at the expected ratio without a developmental defect, unlike previously reported Brca1 mutant mice. However, Brca1(S971A/S971A) mice suffered a moderately increased risk of spontaneous tumor formation, with a majority of females developing uterus hyperplasia and ovarian abnormalities by 2 years of age. After treatment with DNA-damaging agents, Brca1(S971A/S971A) mice exhibited several abnormalities, including increased body weight, abnormal hair growth pattern, lymphoma, mammary tumors, and endometrial tumors. In addition, the onset of tumor formation became accelerated, and 80% of the mutant mice had developed tumors by 1 year of age. We demonstrated that the Brca1(S971A/S971A) cells displayed reduced ability to activate the G(2)/M cell cycle checkpoint upon gamma-irradiation and to stabilize p53 following N-methyl-N'-nitro-N-nitrosoguanidine treatment. These observations suggest that Chk2 phosphorylation of S971 is involved in Brca1 function in modulating the DNA damage response and repressing tumor formation.

Characterization of the UDP-N-acetylgalactosamine binding domain of bovine polypeptide alphaN-acetylgalactosaminyltransferase T1.

UDP-GalNAc:polypeptide alphaN-acetylgalactosaminyltransferases (ppGaNTases) transfer GalNAc from UDP-GalNAc to Ser or Thr. Structural features underlying their enzymatic activity and their specificity are still unidentified. In order to get some insight into the donor substrate recognition, we used a molecular modelling approach on a portion of the catalytic site of the bovine ppGaNTase-T1. Fold recognition methods identified as appropriate templates the bovine alpha1,3galactosyltransferase and the human alpha1,3N-acetylgalactosaminyltransferase. A model of the ppGaNTase-T1 nucleotide-sugar binding site was built into which the UDP-GalNAc and the Mn2+ cation were docked. UDP-GalNAc fits best in a conformation where the GalNAc is folded back under the phosphates and is maintained in that special conformation through hydrogen bonds with R193. The ribose is found in van der Waals contacts with F124 and L189. The uracil is involved in a stacking interaction with W129 and forms a hydrogen bond with N126. The Mn2+ is found in coordination both with the phosphates of UDP and the DXH motif of the enzyme. Amino acids in contact with UDP-GalNAc in the model have been mutated and the corresponding soluble forms of the enzyme expressed in yeast. Their kinetic constants confirm the importance of these amino acids in donor substrate interactions.

Nuclear receptor LXRalpha is involved in cAMP-mediated human renin gene expression.

The cAMP-signaling pathway plays a crucial role in the regulation of the renin gene, but the mechanism involved remains poorly understood. We have focused our studies of renin gene regulation on the unique cAMP responsive element (huREN/CNRE, -135 to -107) in the human renin promoter. We have cloned a protein that binds to this unique CNRE and demonstrated that this protein is liver X receptor-alpha (LXRalpha), a transcriptional factor of the nuclear receptor family. Transient expression of LXRalpha in human renin-producing Calu-6 cells increased cAMP inducibility of human renin promoter. Similarly, LXRalpha-stably transfected Calu-6 cells exhibited increased cAMP inducibility of renin promoter as well as the endogenous renin gene. Site-directed mutation of huREN/CNRE, which disrupted LXRalpha binding, decreased cAMP-induced transcriptional activity of human renin promoter. Furthermore, we demonstrated that the binding of LXRalpha derived from human juxtaglomerular cells, the main production site of renin in the kidney, to the huREN/CNRE in vivo. These results suggest that LXRalpha plays an important role in the cAMP-mediated regulation of human renin gene transcription by binding to CNRE.

C-terminal domain of insulin-like growth factor (IGF) binding protein-6: structure and interaction with IGF-II.

IGFs are important mediators of growth. IGF binding proteins (IGFBPs) 1-6 regulate IGF actions and have IGF-independent actions. The C-terminal domains of IGFBPs contribute to high-affinity IGF binding and modulation of IGF actions and confer some IGF-independent properties, but understanding how they achieve this has been constrained by the lack of a three-dimensional structure. We therefore determined the solution structure of the C-domain of IGFBP-6 using nuclear magnetic resonance (NMR). The domain consists of a thyroglobulin type 1 fold comprising an alpha-helix followed by a loop, a three-stranded antiparallel beta-sheet incorporating a second loop, and finally a disulfide-bonded flexible third loop. The IGF-II binding site on the C-domain was identified by examining NMR spectral changes upon complex formation. It consists of a largely hydrophobic surface patch involving the alpha-helix, the first beta-strand, and the first and second loops. The site was confirmed by mutagenesis of several residues, which resulted in decreased IGF binding affinity. The IGF-II binding site lies adjacent to surfaces likely to be involved in glycosaminoglycan binding of IGFBPs, which might explain their decreased IGF affinity when bound to glycosaminoglycans, and nuclear localization. Our structure provides a framework for understanding the roles of IGFBP C-domains in modulating IGF actions and conferring IGF-independent actions, as well as ultimately for the development of therapeutic IGF inhibitors for diseases including cancer.

Mutational analysis of histidine residues in human organic anion transporter 4 (hOAT4).

Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, antitumour drugs, antibiotics, anti-hypertensives and anti-inflammatories. hOAT4-mediated transport of the organic anion oestrone sulphate in COS-7 cells was inhibited by the histidine-modifying reagent DEPC (diethyl pyrocarbonate). Therefore the role of histidine residues in the function of hOAT4 was examined by site-directed mutagenesis. All five histidine residues of hOAT4 were converted into alanine, singly or in combination. Single replacement of His-47, or simultaneous replacement of His-47/52/83 or His-47/52/83/305/469 (H-less) led to a 50-80% decrease in transport activity. The decreased transport activity of these mutants was correlated with a decreased amount of cell-surface expression, although the total cell expression of these mutants was similar to that of wild-type hOAT4. These results suggest that mutation at positions 47, 47/52/83 and 47/52/83/305/469 impaired membrane expression rather than function. We also showed that, although most of the histidine mutants of hOAT4 were sensitive to inhibition by DEPC, H469A (His-469-->Ala) was completely insensitive to inhibition by this reagent. Therefore modification of His-469 is responsible for the inhibition of hOAT4 by DEPC.

Nuclear factor of activated T cells (NFAT) is involved in the depolarization-induced activation of growth hormone-releasing hormone gene transcription in vitro.

GHRH plays a pivotal role in the regulation of both synthesis and secretion of GH in the anterior pituitary. In this study, we examined the molecular mechanism of depolarization-induced GHRH gene transcription using the hypothalamus cell line, Gsh+/+, revealing the involvement of the transcription factor called nuclear factor of activated T cells (NFAT). GHRH, NFAT1, NFAT4, and related genes were endogenously expressed in Gsh+/+ cells and the rat arcuate nucleus, where NFAT1 and GHRH were colocalized. Cellular excitation with high potassium potently stimulated endogenous GHRH gene 5'-promoter activity as well as the NFAT-mediated gene transcription, the former being further enhanced by coexpression of NFAT. On the other hand, cyclosporin A (a calcineurin-NFAT inhibitor) or EGTA (a calcium chelator) significantly blocked the depolarization-induced GHRH gene transcription. EMSA and site-directed mutagenesis experiments showed the direct binding of NFAT at five sites of the GHRH promoter, among which the relative importance of three distal sites (-417/-403, -402/-387, -317/-301) was suggested. Finally, elimination of all five sites completely abolished the NFAT-induced GHRH gene up-regulation. Altogether, our results suggest that the transcription factor NFAT is involved in the depolarization-induced transcriptional activation of GHRH gene in the neuronal cells.

Phosphorylation and binding interactions of CheY studied by use of Badan-labeled protein.

In the chemotaxis signal transduction pathway of Escherichia coli, the response regulator protein CheY is phosphorylated by the receptor-coupled protein kinase CheA. Previous studies of CheY phosphorylation and CheY interactions with other proteins in the chemotaxis pathway have exploited the fluorescence properties of Trp(58), located immediately adjacent to the phosphorylation site of CheY (Asp(57)). Such studies can be complicated by the intrinsic fluorescence and absorbance properties of CheA and other proteins of interest. To circumvent these difficulties, we generated a derivative of CheY carrying a covalently attached fluorescent label that serves as a sensitive reporter of phosphorylation and binding events and that absorbs and emits light at wavelengths well removed from potential interference by other proteins. This labeled version of CheY has the (dimethylamino)naphthalene fluorophore from Badan [6-bromoacetyl-2-(dimethylamino)naphthalene] attached to the thiol group of a cysteine introduced at position 17 of CheY by site-directed mutagenesis. Under phosphorylating conditions (or in the presence of beryllofluoride), the fluorescence emission of Badan-labeled CheY(M17C) exhibited an approximately 10 nm blue shift and an approximately 30% increase in signal intensity at 490 nm. The fluorescence of Badan-labeled CheY(M17C) also served as a sensitive reporter of CheY-CheA binding interactions, exhibiting an approximately 50% increase in emission intensity in the presence of saturating levels of CheA. Compared to wild-type CheY, Badan-labeled CheY exhibited reduced ability to autodephosphorylate and could not interact productively with the phosphatase CheZ. However, with respect to autophosphorylation and interactions with CheA, Badan-CheY performed identically to wild-type CheY, allowing us to explore CheA-CheY phosphotransfer kinetics and binding kinetics without interference from the fluorescence/absorbance properties of CheA and ATP. These results provide insights into CheY interactions with CheA, CheZ, and other components of the chemotaxis signaling pathway.

Disruption of dimerization and substrate phosphorylation inhibit factor inhibiting hypoxia-inducible factor (FIH) activity.

HIF (hypoxia-inducible factor) is an alphabeta transcription factor that modulates the hypoxic response in many animals. The cellular abundance and activity of HIF-alpha are regulated by its post-translational hydroxylation. The hydroxylation of HIF is catalysed by PHD (prolyl hydroxylase domain) enzymes and FIH (factorinhibiting HIF), all of which are 2-oxoglutarate- and Fe(II)-dependent dioxygenases. FIH hydroxylates a conserved asparagine residue in HIF-alpha (Asn-803), which blocks the binding of HIF to the transcriptional co-activator p300, preventing transcription of hypoxia-regulated genes under normoxic conditions. In the present paper, we report studies on possible mechanisms for the regulation of FIH activity. Recently solved crystal structures of FIH indicate that it is homodimeric. Site-directed mutants of FIH at residues Leu-340 and Ile-344, designed to disrupt dimerization, were generated in order to examine the importance of the dimeric state in determining FIH activity. A single point mutant, L340R (Leu-340-->Arg), was shown to be predominantly monomeric and to have lost catalytic activity as measured by assays monitoring 2-oxoglutarate turnover and asparagine hydroxylation. In contrast, the I344R (Ile-344-->Arg) mutant was predominantly dimeric and catalytically active. The results imply that the homodimeric form of FIH is required for productive substrate binding. The structural data also revealed a hydrophobic interaction formed between FIH and a conserved leucine residue (Leu-795) on the HIF substrate, which is close to the dimer interface. A recent report has revealed that phosphorylation of Thr-796, which is adjacent to Leu-795, enhances the transcriptional response in hypoxia. Consistent with this, we show that phosphorylation of Thr-796 prevents the hydroxylation of Asn-803 by FIH.

Electrostatic interactions of domain III stabilize the inactive conformation of mu-calpain.

The ubiquitous mu- and m-calpains are Ca2+-dependent cysteine proteases. They are activated via rearrangement of the catalytic domain II induced by cooperative binding of Ca2+ to several sites of the molecule. Based on the crystallographic structures, a cluster of acidic residues in domain III, the acidic loop, has been proposed to function as part of an electrostatic switch in the activation process. Experimental support for this hypothesis was obtained by site-directed mutagenesis of recombinant human mu-calpain expressed with the baculovirus system in insect cells. Replacing the acidic residues of the loop individually with alanine resulted in an up to 7-fold reduction of the half-maximal Ca2+ concentration required for conformational changes (probed with 2-p-toluidinylnapthalene-6-sulphonate fluorescence) and for enzymic activity. Along with structural information, the contribution of individual acidic residues to the Ca2+ requirement for activation revealed that interactions of the acidic loop with basic residues in the catalytic subdomain IIb and in the pre-transducer region of domain III stabilize the structure of inactive micro-calpain. Disruption of these electrostatic interactions makes the molecule more flexible and increases its Ca2+ sensitivity. It is proposed that the acidic loop and the opposing basic loop of domain III constitute a double-headed electrostatic switch controlling the assembly of the catalytic domain.

Heterodimeric interaction and interfaces of S100A1 and S100P.

With the widespread use of yeast two-hybrid systems, many heterodimeric forms of S100 proteins have been found, although their biological significance is unknown. In the present study, S100A1 was found to interact with another S100 protein, S100P, by using the yeast two-hybrid system. The binding parameters of the interaction were obtained using an optical biosensor and show that S100P has a slightly higher affinity for S100A1 (K(d)=10-20 nM) when compared with that for self-association (K(d)=40-120 nM). The physical interaction of S100A1 and S100P was also demonstrated in living mammalian cells using a fluorescence resonance energy transfer technique. Preincubation of recombinant S100P with S100A1, before the biosensor assay, reduced by up to 50% the binding of S100P to a recombinant C-terminal fragment of non-muscle myosin A, one of its target molecules. Site-specific mutations of S100P and S100A1, combined with homology modelling of an S100P/S100A1 heterodimer using known S100P and S100A1 structures, allowed the hydrophobic interactions at the dimeric interface of the heterodimer to be defined and provide an explanation for the heterodimerization of S100P and S100A1 at the molecular level. These results have revealed the similarities and the differences between the S100P homodimer and the S100A1/S100P heterodimer.

Gln212, Asn270, and Arg301 are critical for catalysis by adenylosuccinate lyase from Bacillus subtilis.

In adenylosuccinate lyase from Bacillus subtilis, Gln(212), Asn(270), and Arg(301) are conserved and located close to the succinyl moiety of docked adenylosuccinate. We constructed mutant enzymes with Gln(212) replaced by Glu and Met, Asn(270) by Asp and Leu, and Arg(301) by Gln or Lys. The wild-type and mutant enzymes were expressed in Escherichia coli and purified to homogeneity. The specific activities of the Q212M and the 270 and 301 mutant enzymes were decreased more than 3000-fold as compared to the wild type. Only Q212E retained sufficient activity for determination of its kinetic parameters: V(max) was decreased approximately 1000-fold, and K(m) was increased 6-fold, as compared to the wild-type enzyme. Adenylosuccinate binding studies of the other mutants revealed greatly weakened affinities that contributed to, but did not account entirely for, the loss of activity. These mutant enzymes did not differ greatly from the wild-type enzyme in secondary structure or subunit association state, as shown by circular dichroism spectroscopy and light-scattering photometry. Incubation of pairs of inactive mutant enzymes led to reconstitution of some functional sites by subunit complementation, with recovery of up to 25% of the specific activity of the wild-type enzyme. Subunit complementation occurs only if the two mutations are contributed to the active site by different subunits. Thus, mixing Q212E with N270L enzyme yielded a specific activity of approximately 20% of the wild-type enzyme, while mixing Q212M with R301K enzyme did not restore activity. As supported by computer modeling, the studies presented here indicate that Gln(212), Asn(270), and Arg(301) are indispensable to catalysis by adenylosuccinate lyase and probably interact noncovalently with the carboxylate anions of the substrates 5-aminoimidazole-4(N-succinylocarboxamide)ribonucleotide and adenylosuccinate, optimizing their bound orientations.