Biomedical subjects

P L Davies

Publications and source records attributed to P L Davies.

At least 19 recordsLinked to original sources

PubMed2006

Calpain inhibition by alpha-ketoamide and cyclic hemiacetal inhibitors revealed by X-ray crystallography.

Calpains are intracellular calcium-activated cysteine proteases whose unregulated proteolysis following the loss of calcium homeostasis can lead to acute degeneration during ischemic episodes and trauma, as well as Alzheimer's disease and cataract formation. The determination of the crystal structure of the proteolytic core of mu-calpain (muI-II) in a calcium-bound active conformation has made structure-guided design of active site inhibitors feasible. We present here high-resolution crystal structures of rat muI-II complexed with two reversible calpain-specific inhibitors employing cyclic hemiacetal (SNJ-1715) and alpha-ketoamide (SNJ-1945) chemistries that reveal new details about the interactions of inhibitors with this enzyme. The SNJ-1715 complex confirms that the free aldehyde is the reactive species of the cornea-permeable cyclic hemiacetal. The alpha-ketoamide warhead of SNJ-1945 binds with the hydroxyl group of the tetrahedral adduct pointing toward the catalytic histidine rather than the oxyanion hole. The muI-II-SNJ-1945 complex shows residue Glu261 displaced from the S1' site by the inhibitor, resulting in an extended "open" conformation of the domain II gating loop and an unobstructed S1' site. This conformation offers an additional template for structure-based drug design extending to the primed subsites. An important role for the highly conserved Glu261 is proposed.

Amino Acid Sequence↗

PubMed2004

Crystal structures of calpain-E64 and -leupeptin inhibitor complexes reveal mobile loops gating the active site.

The endogenous calpain inhibitor, calpastatin, modulates some patho-physiological aspects of calpain signaling. Excess calpain can escape this inhibition and as well, many calpain isoforms and autolytically generated protease core fragments are not inhibited by calpastatin. There is a need, therefore, to develop specific, cell-permeable calpain inhibitors to block uncontrolled proteolysis and prevent tissue damage during brain and heart ischemia, spinal-cord injury and Alzheimer's diseases. Here, we report the first high-resolution crystal structures of rat mu-calpain protease core complexed with two traditional, low molecular mass inhibitors, leupeptin and E64. These structures show that access to a slightly deeper, but otherwise papain-like active site is gated by two flexible loops. These loops are divergent among the calpain isoforms giving a potential structural basis for substrate/inhibitor selectivity over other papain-like cysteine proteases and between members of the calpain family.

Amino Acid Sequence↗

PubMed2003

A second binding site revealed by C-terminal truncation of calpain small subunit, a penta-EF-hand protein.

The subunits in calpain and in the related penta-EF-hand (PEF) proteins are bound through contacts between the unpaired EF-hand 5 from each subunit. To study subunit binding further, a tetra-EF-hand 18 kDa N- and C-terminally truncated form of the calpain small subunit was prepared (18k). This protein does not combine with the calpain large subunit to form active calpain, but forms homodimers in solution, as shown by ultracentrifugation. The X-ray structure of the 18k protein in the presence of cadmium was solved to a resolution of 2.0 A. The structure of the monomer is almost identical to the known structure of the calpain small subunit, but the 18k protein forms an oligomer in the crystal by the use of two binding sites. One of these sites is an artefact arising from the C-terminal truncation, but the other is a naturally occurring site that is fully exposed to water in intact purified calpain. The characteristics of this site suggest that it may be important in binding other protein modulators involved in the regulation of calpain and of PEF proteins.

Binding Sites↗

PubMed2001

Characterization and cloning of a Tenebrio molitor hemolymph protein with sequence similarity to insect odorant-binding proteins.

The yellow mealworm beetle, Tenebrio molitor, produces a number of moderately abundant low molecular weight hemolymph proteins ( approximately 12 kDa) which behave in a similar manner during purification and share antigenic epitopes. The cDNA sequence of the major component (THP12) was determined and the deduced protein sequence was found to be similar to those of insect odorant-binding proteins. Southern blot analysis suggests that at least some of the diversity in this family of proteins is encoded at the gene level. Both northern and western blot analysis indicate that THP12 is present in a variety of developmental stages and both sexes. THP12 was originally classified as an antifreeze protein, but the lack of antifreeze activity in the recombinant protein, as well as the clear separation of the antifreeze activity from THP12 following HPLC purification, has ruled out this function. The abundance of THP12, the similarity of THP12 to insect odorant-binding proteins, and the presence of hydrophobic cavities inside the protein (Rothemund et al., A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands. Structure, 7 (1999) 1325-1332.) suggest that THP12 may function to carry non-water soluble compounds in the hemolymph. THP12 is also similar, particularly in structurally important regions, to other insect proteins from non-sensory tissues, suggesting the existence of a large family of carrier proteins which may perform diverse functions throughout the insect.

Amino Acid Sequence↗

PubMed2001

Ca(2+)-induced structural changes in rat m-calpain revealed by partial proteolysis.

Partial proteolysis by exogenous proteases in the presence and absence of Ca(2+) was used to map the protease-resistant domains in m-calpain, and to obtain evidence for the conformational changes induced in this thiol protease by Ca(2+). The complication of autoproteolysis was avoided by using the inactive Cys105Ser calpain mutant. Both trypsin and chymotrypsin produced similar cleavage patterns from the large subunit (domains I-IV), while the small subunit (domain VI) was largely unaffected. N-Terminal sequencing of the major products showed that hydrolysis occurred in the N-terminal anchor peptide, which binds domain I to domain VI, at a site close to the C terminus of domain II, and at several sites within domain III. Of particular importance to the overall Ca(2+)-induced conformational changes was the increase in mobility and accessibility of domain III. The same sites were cleaved in the presence and absence of Ca(2+), but with one exception digestion was much more rapid in the presence of Ca(2+). The exception was a site close to residue 255 located within the active site cleft. This site was accessible to cleavage in the absence of Ca(2+), when the active site is not assembled, but was protected in the presence of Ca(2+). This result supports the hypothesis that Ca(2+) induces movement of domains I and II closer together to form the functional active site of calpain.

Amino Acid Sequence↗

PubMed2001

Structure of type I antifreeze protein and mutants in supercooled water.

Many organisms are able to survive subzero temperatures at which bodily fluids would normally be expected to freeze. These organisms have adapted to these lower temperatures by synthesizing antifreeze proteins (AFPs), capable of binding to ice, which make further growth of ice energetically unfavorable. To date, the structures of five AFPs have been determined, and they show considerable sequence and structural diversity. The type I AFP reveals a single 37-residue alpha-helical structure. We have studied the behavior of wild-type type I AFP and two "inactive" mutants (Ala17Leu and Thr13Ser/Thr24Ser) in normal and supercooled solutions of H(2)O and deuterium oxide (D(2)O) to see if the structure at temperatures below the equilibrium freezing point is different from the structure observed at above freezing temperatures. Analysis of 1D (1)H- and (13)C-NMR spectra illustrate that all three proteins remain folded as the temperature is lowered and even seem to become more alpha-helical as evidenced by (13)C(alpha)-NMR chemical shift changes. Furthermore, (13)C-T(2) NMR relaxation measurements demonstrate that the rotational correlation times of all three proteins behave in a predictable manner under all temperatures and conditions studied. These data have important implications for the structure of the AFP bound to ice as well as the mechanisms for ice-binding and protein oligomerization.

Animals↗

PubMed2001

A theoretical model of a plant antifreeze protein from Lolium perenne.

Antifreeze proteins (AFPs), found in certain organisms enduring freezing environments, have the ability to inhibit damaging ice crystal growth. Recently, the repetitive primary sequence of the AFP of perennial ryegrass, Lolium perenne, was reported. This macromolecular antifreeze has high ice recrystallization inhibition activity but relatively low thermal hysteresis activity. We present here a theoretical three-dimensional model of this 118-residue plant protein based on a beta-roll domain with eight loops of 14-15 amino acids. The fold is supported by a conserved valine hydrophobic core and internal asparagine ladders at either end of the roll. Our model, which is the first proposed for a plant AFP, displays two putative, opposite-facing, ice-binding sites with surface complementarity to the prism face of ice. The juxtaposition of the two imperfect ice-binding surfaces suggests an explanation for the protein's inferior thermal hysteresis but superior ice recrystallization inhibition activity and activity when compared with fish and insect AFPs.

Amino Acid Motifs↗

PubMed2001

Mutations in calpain 3 associated with limb girdle muscular dystrophy: analysis by molecular modeling and by mutation in m-calpain.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder characterized by selective atrophy of the proximal limb muscles. Its occurrence is correlated, in a large number of patients, with defects in the human CAPN3 gene, a gene that encodes the skeletal muscle-specific member of the calpain family, calpain 3 (or p94). Because calpain 3 is difficult to study due to its rapid autolysis, we have developed a molecular model of calpain 3 based on the recently reported crystal structures of m-calpain and on the high-sequence homology between p94 and m-calpain (47% sequence identity). On the basis of this model, it was possible to explain many LGMD2A point mutations in terms of calpain 3 inactivation, supporting the idea that loss of calpain 3 activity is responsible for the disease. The majority of the LGMD2A mutations appear to affect domain/domain interaction, which may be critical in the assembly and the activation of the multi-domain calpain 3. In particular, we suggest that the flexibility of protease domain I in calpain 3 may play a critical role in the functionality of calpain 3. In support of the model, some clinically observed calpain 3 mutations were generated and analyzed in recombinant m-calpain. Mutations of residues forming intramolecular domain contacts caused the expected loss of activity, but mutations of some surface residues had no effect on activity, implying that these residues in calpain 3 may interact in vivo with other target molecules. These results contribute to an understanding of structure-function relationships and of pathogenesis in calpain 3.

Amino Acid Sequence↗

PubMed2001

Error-negativity and positivity as they relate to other ERP indices of attentional control and stimulus processing.

We compared individual differences in the ERP associated with incorrect responses in a discrimination task with other ERP components associated with attentional control and stimulus discrimination (N2, P3, CNV). Trials with errors that are detected by the subject normally produce a negativity (N(E)) immediately following the response followed by a positivity (P(E)). The morphology of the N(E) and the P(E) is similar to that of the standard N2-P3 complex on correct discrimination trials. Our findings suggest that the P(E) is a P3 response to the internal detection of errors. The N(E), however, appears to be distinct from the N2. Finally, even though both the contingent negative variation (CNV) and the N(E) are associated with prefrontal cortex and the allocation of attention to response accuracy, the N(E) and CNV did not relate to one another.

Adult↗

PubMed2001

Sialic acid synthase: the origin of fish type III antifreeze protein?

Fish type III antifreeze protein is homologous to the C-terminal region of mammalian sialic acid synthase. Similarity is greatest in the protein core and the flat ice-binding region. This relationship adds to the growing list of links between ice-binding proteins (antifreezes) and proteins that interact with sugars and polysaccharides.

Amino Acid Sequence↗

PubMed2001

Antifreeze protein from shorthorn sculpin: identification of the ice-binding surface.

Shorthorn sculpins, Myoxocephalus scorpius, are protected from freezing in icy seawater by alanine-rich, alpha-helical antifreeze proteins (AFPs). The major serum isoform (SS-8) has been reisolated and analyzed to establish its correct sequence. Over most of its length, this 42 amino acid protein is predicted to be an amphipathic alpha-helix with one face entirely composed of Ala residues. The other side of the helix, which is more heterogeneous and hydrophilic, contains several Lys. Computer simulations had suggested previously that these Lys residues were involved in binding of the peptide to the [11-20] plane of ice in the <-1102> direction. To test this hypothesis, a series of SS-8 variants were generated with single Ala to Lys substitutions at various points around the helix. All of the peptides retained significant alpha-helicity and remained as monomers in solution. Substitutions on the hydrophilic helix face at position 16, 19, or 22 had no obvious effect, but those on the adjacent Ala-rich surface at positions 17, 21, and 25 abolished antifreeze activity. These results, with support from our own modeling and docking studies, show that the helix interacts with the ice surface via the conserved alanine face, and lend support to the emerging idea that the interaction of fish AFPs with ice involves appreciable hydrophobic interactions. Furthermore, our modeling suggests a new N terminus cap structure, which helps to stabilize the helix, whereas the role of the lysines on the hydrophilic face may be to enhance solubility of the protein.

Alanine↗

PubMed2001

Antifreeze proteins of teleost fishes.

Marine teleosts at high latitudes can encounter ice-laden seawater that is approximately 1 degrees C colder than the colligative freezing point of their body fluids. They avoid freezing by producing small antifreeze proteins (AFPs) that adsorb to ice and halt its growth, thereby producing an additional non-colligative lowering of the freezing point. AFPs are typically secreted by the liver into the blood. Recently, however, it has become clear that AFP isoforms are produced in the epidermis (skin, scales, fin, and gills) and may serve as a first line of defense against ice propagation into the fish. The basis for the adsorption of AFPs to ice is something of a mystery and is complicated by the extreme structural diversity of the five antifreeze types. Despite the recent acquisition of several AFP three-dimensional structures and the definition of their ice-binding sites by mutagenesis, no common ice-binding motif or even theme is apparent except that surface-surface complementarity is important for binding. The remarkable diversity of antifreeze types and their seemingly haphazard phylogenetic distribution suggest that these proteins might have evolved recently in response to sea level glaciation occurring just 1-2 million years ago in the northern hemisphere and 10-30 million years ago around Antarctica. Not surprisingly, the expression of AFP genes from different origins can also be quite dissimilar. The most intensively studied system is that of the winter flounder, which has a built-in annual cycle of antifreeze expression controlled by growth hormone (GH) release from the pituitary in tune with seasonal cues. The signal transduction pathway, transcription factors, and promoter elements involved in this process are just beginning to be characterized.

Animals↗

PubMed2000

Calpain mutants with increased Ca2+ sensitivity and implications for the role of the C(2)-like domain.

The ubiquitous calpain isoforms (mu- and m-calpain) are Ca(2+)-dependent cysteine proteases that require surprisingly high Ca(2+) concentrations for activation in vitro ( approximately 50 and approximately 300 microm, respectively). The molecular basis of such a high requirement for Ca(2+) in vitro is not known. In this study, we substantially reduced the concentration of Ca(2+) required for the activation of m-calpain in vitro through the specific disruption of interdomain interactions by structure-guided site-directed mutagenesis. Several interdomain electrostatic interactions involving lysine residues in domain II and acidic residues in the C(2)-like domain III were disrupted, and the effects of these mutations on activity and Ca(2+) sensitivity were analyzed. The mutation to serine of Glu-504, a residue that is conserved in both mu- and m-calpain and interacts most notably with Lys-234, reduced the in vitro Ca(2+) requirement for activity by almost 50%. The mutation of Lys-234 to serine or glutamic acid resulted in a similar reduction. These are the first reported cases in which point mutations have been able to reduce the Ca(2+) requirement of calpain. The structures of the mutants in the absence of Ca(2+) were shown by x-ray crystallography to be unchanged from the wild type, demonstrating that the increase in Ca(2+) sensitivity was not attributable to conformational change prior to activation. The conservation of sequence between mu-calpain, m-calpain, and calpain 3 in this region suggests that the results can be extended to all of these isoforms. Whereas the primary Ca(2+) binding is assumed to occur at EF-hands in domains IV and VI, these results show that domain II-domain III salt bridges are important in the process of the Ca(2+)-induced activation of calpain and that they influence the overall Ca(2+) requirement of the enzyme.

Animals↗

PubMed2000

Mimicry of ice structure by surface hydroxyls and water of a beta-helix antifreeze protein.

Insect antifreeze proteins (AFP) are much more effective than fish AFPs at depressing solution freezing points by ice-growth inhibition. AFP from the beetle Tenebrio molitor is a small protein (8.4 kDa) composed of tandem 12-residue repeats (TCTxSxxCxxAx). Here we report its 1.4-A resolution crystal structure, showing that this repetitive sequence translates into an exceptionally regular beta-helix. Not only are the 12-amino-acid loops almost identical in the backbone, but also the conserved side chains are positioned in essentially identical orientations, making this AFP perhaps the most regular protein structure yet observed. The protein has almost no hydrophobic core but is stabilized by numerous disulphide and hydrogen bonds. On the conserved side of the protein, threonine-cysteine-threonine motifs are arrayed to form a flat beta-sheet, the putative ice-binding surface. The threonine side chains have exactly the same rotameric conformation and the spacing between OH groups is a near-perfect match to the ice lattice. Together with tightly bound co-planar external water, three ranks of oxygen atoms form a two-dimensional array, mimicking an ice section.

Animals↗

PubMed2000

Beta-helix structure and ice-binding properties of a hyperactive antifreeze protein from an insect.

Insect antifreeze proteins (AFP) are considerably more active at inhibiting ice crystal growth than AFP from fish or plants. Several insect AFPs, also known as thermal hysteresis proteins, have been cloned and expressed. Their maximum activity is 3-4 times that of fish AFPs and they are 10-100 times more effective at micromolar concentrations. Here we report the solution structure of spruce budworm (Choristoneura fumiferana) AFP and characterize its ice-binding properties. The 9-kDa AFP is a beta-helix with a triangular cross-section and rectangular sides that form stacked parallel beta-sheets; a fold which is distinct from the three known fish AFP structures. The ice-binding side contains 9 of the 14 surface-accessible threonines organized in a regular array of TXT motifs that match the ice lattice on both prism and basal planes. In support of this model, ice crystal morphology and ice-etching experiments are consistent with AFP binding to both of these planes and thus may explain the greater activity of the spruce budworm antifreeze.

Animals↗

PubMed2000

Folding and structural characterization of highly disulfide-bonded beetle antifreeze protein produced in bacteria.

The hyperactive antifreeze protein from the beetle, Tenebrio molitor, is an 8.5-kDa, threonine-rich protein containing 16 Cys residues, all of which are involved in disulfide bonds. When produced by Escherichia coli, the protein accumulated in the supernatant in an inactive, unfolded state. Its correct folding required days or weeks of oxidation at 22 or 4 degrees C, respectively, and its purification included the removal of imperfectly folded forms by reversed-phase HPLC. NMR spectroscopy was used to assess the degree of folding of each preparation. One-dimensional (1)H and two-dimensional (1)H total correlation spectroscopy spectra were particularly helpful in establishing the characteristics of the fully folded antifreeze in comparison to less well-folded forms. The recombinant antifreeze had no free -SH groups and was rapidly and completely inactivated by 10 mM DTT. It had a thermal hysteresis activity of 2.5 degrees C at a concentration of 1 mg/ml, whereas fish antifreeze proteins typically show a thermal hysteresis of approximately 1.0 degrees C at 10-20 mg/ml. The circular dichroism spectra of the beetle antifreeze had a superficial resemblance to those of alpha-helical proteins, but deconvolution of the spectra indicated the absence of alpha-helix and the presence of beta-structure and coil. NMR analysis and secondary structure predictions agree with the CD data and are consistent with a beta-helix model proposed for the antifreeze on the basis of its 12-amino-acid repeating structure and presumptive disulfide bond arrangement.

Animals↗

PubMed2000

Structure-function relationships in spruce budworm antifreeze protein revealed by isoform diversity.

The spruce budworm, Choristoneura fumiferana, produces antifreeze protein (AFP) to assist in the protection of the overwintering larval stage. AFPs are thought to lower the freezing point of the hemolymph, noncolligatively, by interaction with the surface of ice crystals. Previously, we had identified a cDNA encoding a 9-kDa AFP with 10-30 times the thermal hysteresis activity, on a molar basis, than that shown by fish AFPs. To identify important residues for ice interaction and to investigate the basis for the hyperactivity of the insect AFPs, six new spruce budworm AFP cDNA isoforms were isolated and sequenced. They differ in amino-acid identity as much as 36% from the originally characterized AFP and can be divided into three classes according to the length of their 3' untranslated regions (UTRs). The new isoforms have at least five putative 'Thr-X-Thr' ice-binding motifs and three of the new isoforms encode larger, 12-kDa proteins. These appear to be a result of a 30 amino-acid insertion bearing two additional ice-binding motifs spaced 15 residues apart. Molecular modeling, based on the NMR structure of a short isoform, suggests that the insertion folds into two additional beta-helix loops with their Thr-X-Thr motifs in perfect alignment with the others. The first Thr of the motifs are often substituted by Val, Ile or Arg and a recombinantly expressed isoform with both Val and Arg substitutions, showed wild-type thermal hysteresis activity. The analysis of these AFP isoforms suggests therefore that specific substitutions at the first Thr in the ice binding motif can be tolerated, and have no discernible effect on activity, but the second Thr appears to be conserved. The second Thr is thus likely important for the dynamics of initial ice contact and interaction by these hyperactive antifreezes.

Amino Acid Motifs↗

PubMed2000

Developmental and environmental regulation of antifreeze proteins in the mealworm beetle Tenebrio molitor.

The yellow mealworm beetle, Tenebrio molitor, contains a family of small Cys-rich and Thr-rich thermal hysteresis proteins that depress the hemolymph freezing point below the melting point by as much as 5. 5 degrees C (DeltaT = thermal hysteresis). Thermal hysteresis protein expression was evaluated throughout development and after exposure to altered environmental conditions. Under favorable growth conditions, small larvae (11-13 mg) had only low levels of thermal hysteresis proteins or thermal hysteresis protein message, but these levels increased 10-fold and 18-fold, respectively, by the final larval instar (> 190 mg), resulting in thermal hysteresis > 3 degrees C. Exposure of small larvae (11-13 mg) to 4 weeks of cold (4 degrees C) caused an approximately 20-fold increase in thermal hysteresis protein concentration, well in excess of the less than threefold developmental increase seen after 4 weeks at 22 degrees C. Exposure of large larvae (100-120 mg) to cold caused 12-fold and sixfold increases in thermal hysteresis protein message and protein levels, respectively, approximately double the maximum levels they would have attained in the final larval instar at 22 degrees C. Thus, thermal hysteresis increased to similar levels (> 4 degrees C) in the cold, irrespective of the size of the larvae (the overwintering stage). At pupation, thermal hysteresis protein message levels decreased > 20-fold and remained low thereafter, but thermal hysteresis activity decreased much more slowly. Exposure to cold did not reverse this decline. Desiccation or starvation of larvae had comparable effects to cold exposure, but surprisingly, short daylength photoperiod or total darkness had no effect on either thermal hysteresis or message levels. As all environmental conditions that caused increased thermal hysteresis also inhibited growth, we postulate that developmental arrest is a primary factor in the regulation of T. molitor thermal hysteresis proteins.

Adaptation, Physiological↗