The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

A peptide from the eel pancreas with structural similarity to human pancreatic secretory trypsin inhibitor

The primary structure of a 61-amino-acid residue peptide from the pancreas of the European eel (Anguilla anguilla) has been established as E E K S G(5)L Y R K P(10)S C G E M(15)S A M H A(20)C P M N F(25)A P V C G(30)T D G N T(35)Y P N E C(40)S L C F Q(45)R Q N T K(50)T D I L I(55)T K D D R(60)C. There was no indication of microheterogeneity. This peptide shows structural similarity to pancreatic secretory trypsin inhibitors from several mammalian species and to a cholecystokinin-releasing peptide isolated from rat pancreatic juice. A comparison of the amino acid sequences of the peptides has identified a domain in the central region of the molecules that has been strongly conserved during evolution. In contrast, the amino acid sequence in the region corresponding to the reactive centre of the mammalian trypsin inhibitors is very poorly conserved in the eel peptide. The P1-P1' reactive site lysine-isoleucine (or arginine-isoleucine) bond in the mammalian trypsin inhibitors is replaced by a methionine-asparagine bond. This region does, however, show limited homology to the reactive centre of human alpha 1-protease inhibitor suggesting that the eel peptide may function as an inhibitor of other proteolytic enzymes in the pancreas.     

Regulation of NADPH Oxidase Activity in Phagocytes: RELATIONSHIP BETWEEN FAD/NADPH BINDING AND OXIDASE COMPLEX ASSEMBLY*

The X⁺-linked chronic granulomatous disease (X⁺-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X⁺-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X⁺-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.     

Interaction of nucleotides with Asp(351) and the conserved phosphorylation loop of sarcoplasmic reticulum Ca(2+)-ATPase.

The nucleotide binding properties of mutants with alterations to Asp(351) and four of the other residues in the conserved phosphorylation loop, (351)DKTGTLT(357), of sarcoplasmic reticulum Ca(2+)-ATPase were investigated using an assay based on the 2', 3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP) photolabeling of Lys(492) and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca(2+), mutations removing the negative charge of Asp(351) (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N(3)-ATP and MgATP with affinities 20-156-fold higher than wild type (K(D) as low as 0.006 microM), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N(3)-ATP 2-1000- and 1-6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys(492). In the absence of Ca(2+), mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg(2+) and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca(2+) (K(D) = 0.001 microM), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca(2+) occlusion due to defective CrATP binding.     

The region from phenylalanine-17 to phenylalanine-28 of a yeast mitochondrial ATPase inhibitor is essential for its ATPase inhibitory activity.

Mitochondrial ATP synthase (F(1)F(o)-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In the present study, we investigated the structure-function relationship of the yeast ATPase inhibitor by amino acid replacement. A total of 22 mutants were isolated and characterized. Five mutants (F17S, R20G, R22G, E25A, and F28S) were entirely inactive, indicating that the residues, Phe17, Arg20, Arg22, Glu25, and Phe28, are essential for the ATPase inhibitory activity of the protein. The activity of 7 mutants (A23G, R30G, R32G, Q36G, L37G, L40S, and L44G) decreased, indicating that the residues, Ala23, Arg30, Arg32, Gln36, Leu37, Leu40, and Leu44, are also involved in the activity. Three mutants, V29G, K34Q, and K41Q, retained normal activity at pH 6.5, but were less active at pH 7.2, indicating that the residues, Val29, Lys34, and Lys41, are required for the protein's action at higher pH. The effects of 6 mutants (D26A, E35V, H39N, H39R, K46Q, and K49Q) were slight or undetectable, and the residues Asp26, Glu35, His39, Lys46, and Lys49 thus appear to be dispensable. The mutant E21A retained normal ATPase inhibitory activity but lacked pH-sensitivity. Competition experiments suggested that the 5 inactivated mutants (F17S, R20G, R22G, E25A, and F28S) could still bind to the inhibitory site on F(1)F(o)-ATPase. These results show that the region from the position 17 to 28 of the yeast inhibitor is the most important for its activity and is required for the inhibition of F(1), rather than binding to the enzyme.     

Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor

The binding of ATP to trimeric P2X receptors (P2XR) causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47–V61 and F324–N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.     

Identification of Functionally Important Residues of the Rat P2X4 Receptor by Alanine Scanning Mutagenesis of the Dorsal Fin and Left Flipper Domains

Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. Here, we focused on the role of these domains in receptor activation, responsiveness to orthosteric ATP analogue agonists, and desensitization. Alanine scanning mutagenesis of the R203-L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor showed that ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists 2-(methylthio)adenosine 5′-triphosphate, adenosine 5′-(γ-thio)triphosphate, 2′(3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate, and α,β-methyleneadenosine 5′-triphosphate. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. Among these mutants, D280A, T281A, R282A, H286A, G291A, and Y292A also exhibited increased time-constant of the desensitizing current response. These experiments, together with homology modeling, indicate that residues located in the upper part of the dorsal fin and left flipper domains, relative to distance from the channel pore, contribute to the organization of the ATP binding pocket and to the initiation of signal transmission towards residues in the lower part of both domains. The R203 and N204 residues, deeply buried in the protein, may integrate the output signal from these two domains towards the gate. In addition, the left flipper residues predominantly account for the control of transition of channels from an open to a desensitized state.     

REVIEWS

Grazing in Terrestrial and Marine Environments (British Ecological Society Symposium No. 4). Edited by D. J. C risp
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Differenciation des Cellules Sexuelles et Eécondation chez les Cryptogames (Protoplasmatologia , Vol. VII/3b). By B. V azart
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Thermophilic Fungi . By D onald G. C ooney and R alph E merson
The Genera of Flowering Flants (Angiospermae). Based Principally on the Genera Plantarum of G. Bentham and J. D. Hooker. Dicotyledones . vol. 1. By J. H utchinson
Algal Cultures and Phytoplankton Ecology . By G. E. F ogg
Flora Europaea, Vol . 1. Eycopodiaceae to Plantanaceae . Edited by T. G. T utin , V. H. H eywood , N. A. B urges , D. H. V alentine , S. M. W alters and D. A. W ebb
The Biology of the Algae . By F. E. R ound
Flora of the British Isles Illustrations . Part 4. Monocotyledons . Drawings by S ybil J. R oles
Plants and the Ecosystem . By W. D. B illings . (Fundamentals of Botany Series, edited by W. A. J ensen and L. G. K avaljian
Essays on Crop Evolution . Edited by S ir J oseph H utchinson     

Functional studies of mutations in the human protoporphyrinogen oxidase gene in variegate porphyria.

The autosomal dominant disorder, variegate porphyria (VP), results from mutations in the protoporphyrinogen oxidase (PPOX) gene. We have investigated the effects of 22 disease-associated missense mutations in this gene on enzyme activity. Mutants were generated in the expression plasmid pHPPOX by site-directed mutagenesis. They were screened for PPOX activity by complementation of the Escherischia coli strain SAS38X which lacks PPOX activity. Ten mutants (G40E, L85P, G232R, de1281H, V282D, L295P, V335G, S350P, L444P, G453V) had no detectable PPOX activity. PPOX activity of the remaining 12 mutants (L15F, R38P, L73P, V84G, D143V, R152C, L154P, V158M, R168H, A172V, V290L, G453R) ranged from less than 1% to 9.2% of wild-type activity. Our findings show that all 22 mutations substantially impair or abolish PPOX activity in a prokaryotic expression system and add to the evidence that they cause VP.     

The sites of phosphorylation of rabbit brain phosphofructo-1-kinase by cyclic AMP-dependent protein kinase

The three isozymic subunits of phosphofructo-1-kinase present in rabbit brain and designated A, B and C were phosphorylated in vitro by cyclic AMP-dependent protein kinase with 32P-labeled ATP. Limited digestion of the labeled enzymes with trypsin or with Staphylococcus aureus V8 proteinase led to the solubilization of radiolabeled peptides derived from the three isozymic subunits. Limited digestion by V8 proteinase was accompanied by a slight reduction in the apparent sizes of the subunits, indicating that the phosphorylated sites are located near either the amino or carboxyl termini of the protein. V8 proteinase digestion led to no change in the maximal activity of the enzyme but did abolish sensitivity to ATP inhibition. The phosphopeptides of the tryptic and the V8 digests were purified by chromatography and their amino acid sequences were determined and compared to the previously established sequence from rabbit muscle isozyme A. PFK-A E H I S R K R S G E A T V PFK-B H V T R R S L S M A K G F PFK-C V S A S P R G S Y R K F L In each instance, the phosphorylated serine, underlined in the above sequences, was found to be one or two residues toward the C-terminus of one or more basic residues. No other similarities in structure were noted.     

Mutants of human αB-crystallin cause enhanced protein aggregation and apoptosis in mammalian cells: Influence of co-expression of HspB1

Mutations of human αB-crystallin cause congenital cataract and cardio-myopathy by protein aggregation and cell death. How mutations of αB-crystallin become pathogenic is poorly understood. To better understand the cellular events related to protein aggregation and cell death, we transfected cataract and cardio-myopathy causing mutants, R11H, P20S, R56W, D109H, R120G, D140N, G154S, R157H and A171T in HeLa cells and assessed protein aggregation and apoptosis by laser scanning confocal microspy (LSCM) and flow cytometry. Cells individually transfected with the mutants, D109H, R120G, D140N and R157H significantly showed more aggregates. Cells overexpressed with HspB1 (Hsp27) significantly sequestered aggregates in all mutants and suppressed apoptosis in mutants, P20S, D109H and A171T. Significant increases of apoptotic cells as stained with Annexin V were observed in mutants, D109H and A171T transfected cells. Cells positive for active caspase-3 was increased in the mutant, D109H. Thus the previously recognized anti-apoptotic functions of αB-crystallin were compromised in these mutants.     

A harmonic structure of the genetic code

In this paper is presented a new, very harmonic structure of the genetic code (GC) within a system of "4 x 5" (and/or of "5 x 4") of amino acids (AAs) in two variants. In first variant, the five rows within the system start with one polar charged amino acid (AA) each, making first column, consisting from five polar charged AAs (D, R, K, H, E). Five polar non-charged AAs (N, P, Y, W, Q) follow, then five non-polar AAs as last column (A, L, F, V, I) and, finally, five polar or non-polar AAs, in a combination, as first to last column (A as non-polar; S, T as polar, and G, P as ambivalent AAs). A second variant is subsequent to this one-"4 x 5" system with five nitrogen AAs (K, R, P, H, W), five oxygen (D, E, Y, S, T), five solely carbon (A, L, F, V, I) and five "combined" AAs (G with hydrogen as side chain; C and M with carbon and sulfur; N and Q with carbon, oxygen and nitrogen). A strict balance of atom and nucleon number as well as molecule mass follows the classification in both system variants.     

Retinitis Pigmentosa Mutants Provide Insight into the Role of the N-terminal Cap in Rhodopsin Folding,Structure, and Function

Autosomal dominant retinitis pigmentosa (ADRP) mutants (T4K, N15S, T17M, V20G, P23A/H/L, and Q28H) in the N-terminal cap of rhodopsin misfold when expressed in mammalian cells. To gain insight into the causes of misfolding and to define the contributions of specific residues to receptor stability and function, we evaluated the responses of these mutants to 11-cis-retinal pharmacological chaperone rescue or disulfide bond-mediated repair. Pharmacological rescue restored folding in all mutants, but the purified mutant pigments in all cases were thermo-unstable and exhibited abnormal photobleaching, metarhodopsin II decay, and G protein activation. As a complementary approach, we superimposed this panel of ADRP mutants onto a rhodopsin background containing a juxtaposed cysteine pair (N2C/D282C) that forms a disulfide bond. This approach restored folding in T4K, N15S, V20G, P23A, and Q28H but not T17M, P23H, or P23L. ADRP mutant pigments obtained by disulfide bond repair exhibited enhanced stability, and some also displayed markedly improved photobleaching and signal transduction properties. Our major conclusion is that the N-terminal cap stabilizes opsin during biosynthesis and contributes to the dark-state stability of rhodopsin. Comparison of these two restorative approaches revealed that the correct position of the cap relative to the extracellular loops is also required for optimal photochemistry and efficient G protein activation.     

Epitope topology and removal of mouse acrosomal plasma membrane by P12-targeted immunoaggregation

P12 is a Kazal-type trypsin inhibitor that has been purified from mouse seminal vesicle secretion. We observed a slight impact of P12 on sperm capacitation, and demonstrated the removal of plasma membrane overlaying the acrosome region by immunoaggregation of P12 on mouse sperm. Further, we compared the immunoreactivity of P12 antibody to ten P12 variants, including six single-site mutated mutants (R19L, Y21V, D22G, R43G, K44S, and R45T), two multisite mutated mutants (R43G/K44S/R45T and L50H/R52G/K53A), and two deletion mutants (Nd10 and Cd8) in which 10 and 8 residues were deleted from the N- and C-terminals, respectively. We found that the N-terminal region, 43RKR45, and the C-terminal region, but not R19, Y21, and D22, are involved in the three epitopes that reside on one side and are three-dimensionally distant from R19, Y21, and D22 on the P12 molecule. Based on the epitope topology, we elucidated the structural basis by which P12 antibody immunoaggregated P12 on sperm head.     

Impact of autosomal recessive juvenile Parkinson's disease mutations on the structure and interactions of the parkin ubiquitin-like domain

Autosomal recessive juvenile parkinsonism (ARJP) is an early onset familial form of Parkinson's disease. Approximately 50% of all ARJP cases are attributed to mutations in the gene park2, coding for the protein parkin. Parkin is a multidomain E3 ubiquitin ligase with six distinct domains including an N-terminal ubiquitin-like (Ubl) domain. In this work we examined the structure, stability, and interactions of the parkin Ubl domain containing most ARJP causative mutations. Using NMR spectroscopy we show that the Ubl domain proteins containing the ARJP substitutions G12R, D18N, K32T, R33Q, P37L, and K48A retained a similar three-dimensional fold as the Ubl domain, while at least one other (V15M) had altered packing. Four substitutions (A31D, R42P, A46P, and V56E) result in poor folding of the domain, while one protein (T55I) showed evidence of heterogeneity and aggregation. Further, of the substitutions that maintained their three-dimensional fold, we found that four of these (V15M, K32T, R33Q, and P37L) lead to impaired function due to decreased ability to interact with the 19S regulatory subunit S5a. Three substitutions (G12R, D18N, and Q34R) with an uncertain role in the disease did not alter the three-dimensional fold or S5a interaction. This work provides the first extensive characterization of the structural effects of causative mutations within the ubiquitin-like domain in ARJP.     

REVIEWS

Book reviewed in this article:
Plant Cells . By F. A. L. C lowes and B. E. J uniper .
The History of British Vegetation . By W inifred P ennington . Modern Biology: General Editor J. E. W ebb .
Flora Europaea , Vol. II. Rosaceae to Umbelliferae . Ed. by T. G. T utin , V. H. H eywood , N. A. B urges , D. M. M oore , D. H. V alentine , S. M. W alters and D. A. W ebb .
Desert Wild Flowers . By E dmund J aeger .
British Mosses and Liverworts . By E. V. W atson .
Anatomy of the Monocotyledons , Vol. III. Commelinales—Zingiberales . By P. B. T omlinson . General Editor C. R. M etcalfe .
Biological Science—The Web of Life . Supervising Editor, D. M organ .
Plant Chimeras . By W. N eilson -J ones .
The Potatoes of Argentine, Brazil, Paraguay and Uruguay, a Biosystematic Study . By J. G. H awkes and J. P. H jerting .
Towards an Understanding of the Mechanism of Heredity . By H. L. K. W hitehouse .
Viruses in Plant Hosts; Form, Distribution and Pathologic Effects . By K atherine E sau .
An Introduction to Industrial Mycology . By G eorge S mith .     

The identification of a domain in Escherichia coli elongation factor Tu that interacts with elongation factor Ts.

A method has been developed to search for the elongation factor Tu (EF-Tu) domain(s) that interact with elongation factor Ts (EF-Ts). This method is based on the suppression of Escherichia coli EF-Tu-dominant negative mutation K136E, a mutation that exerts its effect by sequestering EF-Ts. We have identified nine single-amino acid- substituted suppression mutations in the region 146-199 of EF-Tu. These mutations are R154C, P168L, A174V, K176E, D181G, E190K, D196G, S197F, and I199V. All suppression mutations but one (R154C) significantly affect EF-Tu's ability to interact with EF-Ts under equilibrium conditions. Moreover, with the exception of mutation A174V, the GDP affinity of EF-Tu appears to be relatively unaffected by these mutations. These results suggest that the domain of residues 154 to 199 on EF-Tu is involved in interacting with EF-Ts. These suppression mutations are also capable of suppressing dominant negative mutants N135D and N135I to various degrees. This suggests that dominant negative mutants N135D and N135I are likely to have the same molecular basis as the K136E mutation. The method we have developed in this study is versatile and can be readily adapted to map other regions of EF-Tu. A model of EF-Ts-catalyzed guanine-nucleotide exchange is discussed.     

NMR structure of the p63 SAM domain and dynamical properties of G534V and T537P pathological mutants, identified in the AEC syndrome

The p63 protein is crucial for epidermal development, and its mutations cause the extrodactyly ectodermal dysplasia and cleft lip/palate syndrome. The three-dimensional solution structure of the p63 sterile α-motif (SAM) domain (residues 505–579), a region crucial to explaining the human genetic disease ankyloblepharon-ectodermal dysplasia-clefting syndrome (AEC), has been determined by nuclear magnetic resonance spectroscopy. The structure indicates that the domain is a monomer with the characteristic five-helix bundle topology observed in other SAM domains. It includes five tightly packed helices with an extended hydrophobic core to form a globular and compact structure. The dynamics of the backbone and the global correlation time of the molecule have also been investigated and compared with the dynamical properties obtained through molecular dynamics simulation. Attempts to purify the pathological G534V and T537P mutants, originally identified in AEC, were not successful because of the occurrence of unspecific proteolytic degradation of the mutated SAM domains. Analysis of the structural dynamic properties of the G534V and T537P mutants through molecular dynamics simulation and comparison with the wild type permits detection of differences in the degree of free-dom of individual residues and discussion of the possible causes for the pathology.     

Active site mutations in CheA, the signal-transducing protein kinase of the chemotaxis system in Escherichia coli.

We investigated the functional roles of putative active site residues in Escherichia coli CheA by generating nine site-directed mutants, purifying the mutant proteins, and quantifying the effects of those mutations on autokinase activity and binding affinity for ATP. We designed these mutations to alter key positions in sequence motifs conserved in the protein histidine kinase family, including the N box (H376 and N380), the G1 box (D420 and G422), the F box (F455 and F459), the G2 box (G470, G472, and G474), and the "GT block" (T499), a motif identified by comparison of CheA to members of the GHL family of ATPases. Four of the mutant CheA proteins exhibited no detectable autokinase activity (Kin(-)). Of these, three (N380D, D420N, and G422A) exhibited moderate decreases in their affinities for ATP in the presence or absence of Mg(2+). The other Kin(-) mutant (G470A/G472A/G474A) exhibited wild-type affinity for ATP in the absence of Mg(2+), but reduced affinity (relative to that of wild-type CheA) in the presence of Mg(2+). The other five mutants (Kin(+)) autophosphorylated at rates slower than that exhibited by wild-type CheA. Of these, three mutants (H376Q, D420E, and F455Y/F459Y) exhibited severely reduced k(cat) values, but preserved K(M)(ATP) and K(d)(ATP) values close to those of wild-type CheA. Two mutants (T499S and T499A) exhibited only small effects on k(cat) and K(M)(ATP). Overall, these results suggest that conserved residues in the N box, G1 box, G2 box, and F box contribute to the ATP binding site and autokinase active site in CheA, while the GT block makes little, if any, contribution. We discuss the effects of specific mutations in relation to the three-dimensional structure of CheA and to binding interactions that contribute to the stability of the complex between CheA and Mg(2+)-bound ATP in both the ground state and the transition state for the CheA autophosphorylation reaction.