Ser84 of human renin contributes to the biphasic pH dependence of the renin-angiotensinogen reaction

The pH dependence of the reaction of various renins was investigated using sheep angiotensinogen as a substrate. Human renin showed two separate peaks, but rat and mouse Ren1 renins showed one peak with a shoulder. A comparison of the predicted subsite residues of human renin with those of rat and mouse Ren1 renins revealed that Arg82, Ser84, Thr85, Ala229, and Thr312 are unique in the human sequence. We examined the possible importance of these residues in the unique pH profile of the human renin reaction by replacing these residues with the corresponding residues of rat renin. The replacement of Ser84 of human renin with Gly changed the pH dependence of the reaction to one peak, similarly to rat and mouse Ren1 renins. Other mutant human renins kept two separate peaks, similarly to wild-type human renin. These results indicate that Ser84 of human renin contributes to the biphasic pH dependence of the renin-angiotensinogen reaction.     

Highly potent and specific inhibitors of human renin

We designed aldehyde derivatives of small peptides representing the C-terminal portion of angiotensin I sequence as an inhibitor of human renin. Among compounds that we synthesized, benzyloxycarbonyl (Z)-Phe-His-Leucinal (compound V), Z-Pro-Phe-His-Leucinal (Compound IV) and Z-[3-(1'-naphthyl)Ala]-His-Leucinal (compound VII) markedly inhibited human renin (IC50, 7.5 X 10(-7), 3.2 X 10(-7) and 8.0 X 10(-8) mol/l, respectively). Compound VII was shown to be noncompetitive (Ki = 2.4 X 10(-7) mol/l). It did not inhibit either cathepsin D or pepsin. Compound V had slight or no inhibitory effect at the concentration of 10(-5) mol/l on six animal renins except for monkey and rabbit renins. Results obtained show that these aldehyde compounds are highly selective and species specific inhibitors for human and monkey renins.     

The location of the ligand-binding site of carbohydrate-binding modules that have evolved from a common sequence is not conserved.

Polysaccharide-degrading enzymes are generally modular proteins that contain non-catalytic carbohydrate-binding modules (CBMs), which potentiate the activity of the catalytic module. CBMs have been grouped into sequence-based families, and three-dimensional structural data are available for half of these families. Clostridium thermocellum xylanase 11A is a modular enzyme that contains a CBM from family 6 (CBM6), for which no structural data are available. We have determined the crystal structure of this module to a resolution of 2.1 A. The protein is a beta-sandwich that contains two potential ligand-binding clefts designated cleft A and B. The CBM interacts primarily with xylan, and NMR spectroscopy coupled with site-directed mutagenesis identified cleft A, containing Trp-92, Tyr-34, and Asn-120, as the ligand-binding site. The overall fold of CBM6 is similar to proteins in CBM families 4 and 22, although surprisingly the ligand-binding site in CBM4 and CBM22 is equivalent to cleft B in CBM6. These structural data define a superfamily of CBMs, comprising CBM4, CBM6, and CBM22, and demonstrate that, although CBMs have evolved from a relatively small number of ancestors, the structural elements involved in ligand recognition have been assembled at different locations on the ancestral scaffold.     

Structure and dynamics of the homodimeric dynein light chain km23

km23 (96 residues, 11 kDa) is the mammalian ortholog of Drosophila roadblock, the founding member of LC7/robl/km23 class of dynein light chains. km23 has been shown to be serine-phosphorylated following TGFbeta receptor activation and to bind the dynein intermediate chain in response to such phosphorylation. Here, we report the three-dimensional solution structure of km23, which is shown to be that of a homodimer, similar to that observed for the heterodimeric complex formed between p14 and MP1, two distantly related members of the MglB/robl superfamily, but distinct from the LC8 and Tctex-1 classes of dynein light chains, which also adopt homodimeric structures. The conserved surface residues of km23, including three serine residues, are located predominantly on a single face of the molecule. Adjacent to this face is a large cleft formed by the incomplete overlap of loops from opposite monomers. As shown by NMR relaxation data collected at two fields, several cleft residues are flexible on the ns-ps and ms-mus timescales. Based on these observations, we propose that the patch of conserved residues on the central face of the molecule corresponds to the site at which km23 binds the dynein intermediate chain and that the flexible cleft formed between the overlap of loops from the two monomers corresponds to the site at which km23 binds other partners, such as the TGFbeta type II receptor or Smad2.     

The influence of glycosylation on the fate of renin expressed in Xenopus oocytes

It has been recently reported that, in Xenopus oocytes injected with the mRNA for human renin, this secretory renal glycoprotein acquires phosphomannosyl residues on its asparagine-linked oligosaccharide chains, remains intracellular and undergoes a proteolytic cleavage which removes the prosegment. To understand the influence of glycosylation on the fate of renin in Xenopus oocytes and whether it is specific for human renin, we have expressed human renin and mouse Ren1 renin, which are glycosylated at two and three selected asparagine residues, respectively, and mouse Ren2 renin, which is not glycosylated, in Xenopus oocytes. The majority of human and Ren1 renins remained intracellular and underwent proteolytic cleavage, whereas mouse Ren2 renin was secreted efficiently. When human and Ren1 renins were expressed in oocytes treated with tunicamycin, both were secreted efficiently. A mutant of human renin, which had amino-acid substitutions at both glycosylation sites, was also secreted efficiently, whereas that mutated at one of the two sites was not. These results indicate that the majority of all of the glycosylated renin molecules remain intracellular and undergo proteolytic cleavage, probably due to the acquisition of phosphomannosyl residues, and the human renin remains intracellular if it is only glycosylated at one of the two sites.     

3-D model analysis of the maxilla of infants with lip-jaw-palate clefts]

For the investigation of three-dimensional morphological changes in the maxilla of children with cleft lip and palate, the use of two-dimensional test analysis is inadequate. Since no standardised three-dimensional method has so far been available, a three-dimensional digital, computer-aided procedure was developed to visualize and metrically analyse the growth of the edentulous maxilla of infants with cleft lip and palate. Chronologically consecutive casts of the maxillas (obtained at the ages of one week, and three, six and twelve months) of five children with complete unilateral CLP were measured optically with the instrument Micromeasure 70. Following digitation, the casts were reconstructed in the computer, aligned and superimposed using the Orthosurf program. The distances between the surfaces were then measured; in addition, the surfaces were segmented perpendicular to the alveolar crest at reference points C1, C1', C2, C2' and I. The volumes of the resulting segments were determined and compare with one another. Specially designed software automated the following steps: 1. identification of reference points; 2. alignment of the cloud of points in a system of coordinates, and 3. identification of the alveolar crest. Our initial results show that (1) the new method enables visualization of the extent and direction of morphological changes of the mucosal surface, and (2) reproducible quantification of these changes via the determination of changes in the volume of defined alveolar segments. The three-dimensional analysis presented here permits a comprehensive three-dimensional measurement of the models of the edentulous maxilla of infants with cleft lip and palate.     

The three-dimensional structure of Escherichia coli porphobilinogen deaminase at 1.76-Å resolution

Porphobilinogen deaminase (PBGD) catalyses the polymerization of four molecules of porphobilinogen to form the 1-hydroxymethylbilane, preuroporphyrinogen, a key intermediate in the biosynthesis of tetrapyrroles. The three-dimensional structure of wild-type PBGD from Escherichia coli has been determined by multiple isomorphous replacement and refined to a crystallographic R-factor of 0.188 at 1.76 Å resolution. The polypeptide chain of PBGD is folded into three α/β domains. Domains 1 and 2 have a similar overall topology, based on a five-stranded, mixed β-sheet. These two domains, which are linked by two hinge segments but otherwise make few direct interactions, form an extensive active site cleft at their interface. Domain 3, an open-faced, anti-parallel sheet of three strands, interacts approximately equally with the other two domains. The dipyrromethane cofactor is covalently attached to a cysteine side-chain borne on a flexible loop of domain 3. The cofactor serves as a primer for the assembly of the tetrapyrrole product and is held within the active site cleft by hydrogen-bonds and salt-bridges that are formed between its acetate and propionate side-groups and the polypeptide chain. The structure of a variant of PBGD, in which the methionines have been replaced with selenomethionines, has also been determined. The cofactor, in the native and functional form of the enzyme, adopts a conformation in which the second pyrrole ring (C2) occupies an internal position in the active site cleft. On oxidation, however, this C2 ring of the cofactor adopts a more external position that may correspond approximately to the site of substrate binding and polypyrrole chain elongation. The side-chain of Asp84 hydrogen-bonds the hydrogen atoms of both cofactor pyrrole nitrogens and also potentially the hydrogen atom of the pyrrole nitrogen of the porphobilinogen molecule bound to the proposed substrate binding site. This group has a key catalytic role, possibly in stabilizing the positive charges that develop on the pyrrole nitrogens during the ring-coupling reactions. Possible mechanisms for the processive elongation of the polypyrrole chain involve: accommodation of the elongating chain within the active site cleft, coupled with shifts in the relative positions of domains 1 and 2 to carry the terminal ring into the appropriate position at the catalytic site; or sequential translocation of the elongating polypyrrole chain, attached to the cofactor on domain 3, through the active site cleft by the progressive movement of domain 3 with respect to domains 1 and 2. Other mechanisms are considered although the amino acid sequence comparisons between PBGDs from all species suggest they share the same three-dimensional structure and mechanism of activity. © 1996 Wiley-Liss, Inc.     

Three-dimensional structure of soybean beta-amylase determined at 3.0 A resolution: preliminary chain tracing of the complex with alpha-cyclodextrin.

The three-dimensional structure of a complex of soybean beta-amylase [EC 3.2.1.2] with an inhibitor, alpha-cyclodextrin, has been determined at 3.0 A resolution by X-ray diffraction analysis. Preliminary chain tracing showed that the enzyme folded into large and small domains. The large domain has a (beta alpha)8 super-secondary structure, while the smaller one is formed from two long loops extending from the beta 3 and beta 4 strands of the (beta alpha)8 structure. The interface of the two domains together with shorter loops from the (beta alpha)8 structure form a deep cleft, in which alpha-cyclodextrin binds slightly away from the center. Two maltose molecules also bind in the cleft. One shares a binding site with alpha-cyclodextrin and the other is situated more deeply in the cleft.     

Evidence for cleft closure in actomyosin upon ADP release

Structural insights into the interaction of smooth muscle myosin with actin have been provided by computer-based fitting of crystal structures into three-dimensional reconstructions obtained by electron cryomicroscopy, and by mapping of structural and dynamic changes in the actomyosin complex. The actomyosin structures determined in the presence and absence of MgADP differ significantly from each other, and from all crystallographic structures of unbound myosin. Coupled to a complex movement ( approximately 34 A) of the light chain binding domain upon MgADP release, we observed a approximately 9 degrees rotation of the myosin motor domain relative to the actin filament, and a closure of the cleft that divides the actin binding region of the myosin head. Cleft closure is achieved by a movement of the upper 50 kDa region, while parts of the lower 50 kDa region are stabilized through strong interactions with actin. This model supports a mechanism in which binding of MgATP at the active site opens the cleft and disrupts the interface, thereby releasing myosin from actin.     

A 1-D model to explore the effects of tissue loading and tissue concentration gradients in the revised Starling principle

The recent experiments in Hu et al. (Am J Physiol Heart Circ Physiol 279: H1724-H1736, 2000) and Adamson et al. (J Physiol 557: 889-907, 2004) in frog and rat mesentery microvessels have provided strong evidence supporting the Michel-Weinbaum hypothesis for a revised asymmetric Starling principle in which the Starling force balance is applied locally across the endothelial glycocalyx layer rather than between lumen and tissue. These experiments were interpreted by a three-dimensional (3-D) mathematical model (Hu et al.; Microvasc Res 58: 281-304, 1999) to describe the coupled water and albumin fluxes in the glycocalyx layer, the cleft with its tight junction strand, and the surrounding tissue. This numerical 3-D model converges if the tissue is at uniform concentration or has significant tissue gradients due to tissue loading. However, for most physiological conditions, tissue gradients are two to three orders of magnitude smaller than the albumin gradients in the cleft, and the numerical model does not converge. A simpler multilayer one-dimensional (1-D) analytical model has been developed to describe these conditions. This model is used to extend Michel and Phillips's original 1-D analysis of the matrix layer (J Physiol 388: 421-435, 1987) to include a cleft with a tight junction strand, to explain the observation of Levick (Exp Physiol 76: 825-857, 1991) that most tissues have an equilibrium tissue concentration that is close to 0.4 lumen concentration, and to explore the role of vesicular transport in achieving this equilibrium. The model predicts the surprising finding that one can have steady-state reabsorption at low pressures, in contrast to the experiments in Michel and Phillips, if a backward-standing gradient is established in the cleft that prevents the concentration from rising behind the glycocalyx.     

Experience with the functional cleft lip repair

The first 12 functional cleft lip repairs performed on unselected consecutive patients immediately following the completion of training by the author are presented. Previous reports on this cleft lip repair have shown excellent results but have always been based on patients operated on by the originator of the procedure. This report gives credence to the ease with which a cleft lip repair that gives reproducible good results can be taught and learned even by plastic surgeons with limited experience. It reviews the technical steps of the procedure, which emphasizes wide undermining and release of the orbicularis oris muscle on the lateral side of the cleft to allow redraping and lengthening of the lip skin, step-by-step layered closure of the mucosa, muscle, and skin, and further vertical lengthening of the lip with a Z-plasty skin closure. Three elements that are difficult to achieve or restore with cleft lip revision are evaluated: (1) achievement of a good skin scar, (2) maintenance of the alar-facial groove, and (3) achievement of adequate lip height without sacrificing horizontal lip length. Ten of the 12 patients had a satisfactory scar, 9 patients had a good alar-facial groove, and all patients had a normal-appearing horizontal lip length. Nine patients required secondary surgery; however, in six patients, this included correction of the nasal deformity that was not corrected at the time of cleft lip repair.     

Crystal structure of VC0702 at 2.0 A: conserved hypothetical protein from Vibrio cholerae

VC0702, a conserved hypothetical protein of unknown function from Vibrio cholerae, resides in a three-gene operon containing the MbaA gene that encodes for a GGDEF and EAL domain-containing protein which is involved in regulating formation of the extracellular matrix of biofilms in Vibrio cholerae. The VC0702 crystal structure has been determined at 2.0 A and refined to Rwork = 22.8% and Rfree = 26.3%. VC0702 crystallized in an orthorhombic crystal lattice in the C222(1) space group with dimensions of a = 66.61 A, b = 88.118 A, and c = 118.35 A with a homodimer in the asymmetric unit. VC0702, which forms a mixed alpha + beta three-layered alphabetaalpha sandwich, belongs to the Pfam DUF84 and COG1986 families of proteins. Sequence conservation within the DUF84 and COG1986 families was used to identify a conserved patch of surface residues that define a cleft and potential substrate-binding site in VC0702. The three-dimensional structure of VC0702 is similar to that of Mj0226 from Methanococcus janeschii, which has been identified as a novel NTPase that binds NTP in a deep cleft similarly located to the conserved patch of surface residues that define an analogous cleft in VC0702. Collectively, the data suggest that VC0702 may have a biochemical function that involves NTP binding and phosphatase activity of some kind, and is likely involved in regulation of the signaling pathway that controls biofilm formation and maintenance in Vibrio cholerae.     

Three-dimensional electron microscopy of the reverse gyrase from Sulfolobus tokodaii

Reverse gyrase is a type IA topoisomerase, found in various hyperthermophiles and promotes ATP-dependent positive supercoiling of DNA. Electron microscopy combined with single particle analyses revealed the three-dimensional structure of the DNA-free Sulfolobus tokodaii reverse gyrase and two-dimensional average images of both the protein alone and that complexed with double-stranded DNA. The 23A resolution map exhibited a parallelogrammatic morphology of 110 x 87 x 43A, which is in good agreement with the crystal structure of the Archaeoglobus fulgidus reverse gyrase. The average image of the complex revealed that the monomeric enzyme binds DNA duplex. Together with this average image of the complex, the three-dimensional map implies that, at the beginning of the supercoiling reaction, DNA is bound within a 10-20A wide cleft in the helicase-like domain. We also speculate that DNA may pass through a 20A wide hole at the end of the cleft.     

Structural study of hinge bending in L-arabinose-binding protein

The L-arabinose-binding protein of Escherichia coli is a periplasmic component of the bacterial L-arabinose transport system. The three-dimensional structure of the molecule has been determined by x-ray diffraction and shown to have two globular domains and a connecting hinge. Theoretical study of the flexibility of the hinge using computer simulation showed that the hinge is quite permissive in that only moderate increases in the internal energy are required for opening the cleft where the L-arabinose-binding site is located. In this study, the structural changes that accompany the hinge bending are analyzed. The results show that bending-induced stresses are accommodated by coupled action of covalent and noncovalent forces within the protein molecule. Strains in internal coordinates (bond lengths, bond angles, and torsional angles) are distributed throughout the hinge region after structural relaxation. The pattern of structural changes within a hinge strand upon bending and relaxation depends in large degree on its geometric relationship with the bending axis (e.g. distance and orientation) and the atomic packing of its immediate environment. The distributed structural changes result in a characteristic zigzag pattern for the directional change at each residue in the hinge strands.     

Three-dimensional structure of endo-1,4-beta-xylanase II from Trichoderma reesei: two conformational states in the active site.

The three-dimensional structure of endo-1,4-beta-xylanase II (XYNII) from Trichoderma reesei has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 18.3% at 1.8 A resolution. The 190 amino acid length protein was found to exist as a single domain where the main chain folds to form two mostly antiparallel beta-sheets, which are packed against each other in parallel. The beta-sheet structure is twisted, forming a large cleft on one side of the molecule. The structure of XYNII resembles that of Bacillus 1,3-1,4-beta-glucanase. The cleft is an obvious suggestion for an active site, which has putative binding sites for at least four xylose residues. The catalytic residues are apparently the two glutamic acid residues (Glu86 and Glu177) in the middle of the cleft. One structure was determined at pH 5.0, corresponding to the pH optimum of XYNII. The second structure was determined at pH 6.5, where enzyme activity is reduced considerably. A clear structural change was observed, especially in the position of the side chain of Glu177. The observed conformational change is probably important for the mechanism of catalysis in XYNII.     

The role of RXR-alpha in retinoic acid-induced cleft palate as assessed with the RXR-alpha knockout mouse

Treatment of pregnant mice with retinoic acid (RA) in mid-gestation produces cleft palate and limb defects in the fetuses. RXR-alpha has been previously shown to mediate the teratogenic effects of RA in the limb. In this study, we show that RXR-alpha is also involved in retinoid-induced palatal clefting. Treatment of RXR-alpha knockout mice with a teratogenic dose of RA on gestation day 11 or 12 induces cleft palate at a lower frequency than that seen in wild-type animals.     

Nonsyndromic cleft lip and palate: no evidence of linkage to HLA or factor 13A.

Nonsyndromic cleft lip with or without cleft palate (CLP) is a common craniofacial anomaly, the etiology of which is not known. Population studies have shown that a large proportion of cases occur sporadically. Recently, segregation analyses applied to CLP families have demonstrated that an autosomal dominant/codominant gene(s) may cause clefting in cases. Associations of autosomal dominant CLP and nonsyndromic cleft palate (CP) with HLA and F13A genes on chromosome 6p have been suggested previously. Linkage to these two areas on chromosome 6p were tested in 12 autosomal dominant families with CLP. With a LOD score of -2 or less for exclusion, no evidence of linkage was found to four chromosome 6p markers. Multipoint analysis showed no evidence of a clefting locus in this region spanning 54 cM on chromosome 6p in these CLP families.     

Probing conserved surfaces on PapD

PapD is the periplasmic chaperone required for the assembly of P pili in pyelonephritic strains of Escherichia coli. It consists of two immunoglobulin-like domains bisected by a subunit binding cleft. PapD is the prototype member of a super family of immunoglobulin-like chaperones that work in concert with their respective ushers to assemble a plethora of adhesive organelles including pilus- and non-pilus-associated adhesins. Three highly conserved residue clusters have been shown to play critical roles in the structure and function of PapD, as determined by site-directed mutagenesis. The in vivo stability of the chaperone depended on the formation of a buried salt bridge within the cleft. Residues along the G1 beta strand were required for efficient binding of subunits consistent with the crystal structure of PapD-peptide complexes. Finally, Thr-53, a residue that is part of a conserved band of residues located on the amino-terminal domain surface opposite the subunit binding cleft, was also found to be critical for pilus assembly, but mutations at Thr-53 did not interfere with chaperone-subunit complex formation.     

The pituitary cleft of the rat: An electron microscopic study

The epithelium of the pituitary cleft and its colloid have been studied at the ultrastructural level in normal, adrenalectomized, castrated, dehydrated and lactating adult rats. Both control and experimental animals showed cells in different stages of degradation inside the cleft and mixed with the colloid. These exfoliated cells seems to have originated from the cleft's epithelium or the adjacent granular cells.In adrenalectomized and castrated rats the rostral epithelium of the cleft had large intercellular spaces that in some instances appeared to open directly into the cleft. Occasionally the adenohypophysial canaliculi of the adrenalectomized rats were also seen connecting with the cleft. In lactating and castrated animals dark cells appeared more frequently in the rostral and caudal epithelium of the cleft.The results obtained suggest that the colloid is originated at least in part by involuted pituitary cells. There is also evidence that the follicular cavities of the Pars distalis and the cleft are continuous structures.