Biomedical subjects

Agnieszka Szyk

Publications and source records attributed to Agnieszka Szyk.

9 recordsLinked to original sources

PubMed2006

Crystal structures of human alpha-defensins HNP4, HD5, and HD6.

Six alpha-defensins have been found in humans. These small arginine-rich peptides play important roles in various processes related to host defense, being the effectors and regulators of innate immunity as well as enhancers of adoptive immune responses. Four defensins, called neutrophil peptides 1 through 4, are stored primarily in polymorphonuclear leukocytes. Major sites of expression of defensins 5 and 6 are Paneth cells of human small intestine. So far, only one structure of human alpha-defensin (HNP3) has been reported, and the properties of the intestine defensins 5 and 6 are particularly poorly understood. In this report, we present the high-resolution X-ray structures of three human defensins, 4 through 6, supplemented with studies of their antimicrobial and chemotactic properties. Despite only modest amino acid sequence identity, all three defensins share their tertiary structures with other known alpha- and beta-defensins. Like HNP3 but in contrast to murine or rabbit alpha-defensins, human defensins 4-6 form characteristic dimers. Whereas antimicrobial and chemotactic activity of HNP4 is somewhat comparable to that of other human neutrophil defensins, neither of the intestinal defensins appears to be chemotactic, and for HD6 also an antimicrobial activity has yet to be observed. The unusual biological inactivity of HD6 may be associated with its structural properties, somewhat standing out when compared with other human alpha-defensins. The strongest cationic properties and unique distribution of charged residues on the molecular surface of HD5 may be associated with its highest bactericidal activity among human alpha-defensins.

Amino Acid Sequence↗

PubMed2006

Crystal structure at 1.9A of E. coli ClpP with a peptide covalently bound at the active site.

ClpP, the proteolytic component of the ATP-dependent ClpAP and ClpXP chaperone/protease complexes, has 14 identical subunits organized in two stacked heptameric rings. The active sites are in an interior aqueous chamber accessible through axial channels. We have determined a 1.9 A crystal structure of Escherichia coli ClpP with benzyloxycarbonyl-leucyltyrosine chloromethyl ketone (Z-LY-CMK) bound at each active site. The complex mimics a tetrahedral intermediate during peptide cleavage, with the inhibitor covalently linked to the active site residues, Ser97 and His122. Binding is further stabilized by six hydrogen bonds between backbone atoms of the peptide and ClpP as well as by hydrophobic binding of the phenolic ring of tyrosine in the S1 pocket. The peptide portion of Z-LY-CMK displaces three water molecules in the native enzyme resulting in little change in the conformation of the peptide binding groove. The heptameric rings of ClpP-CMK are slightly more compact than in native ClpP, but overall structural changes were minimal (rmsd approximately 0.5 A). The side chain of Ser97 is rotated approximately 90 degrees in forming the covalent adduct with Z-LY-CMK, indicating that rearrangement of the active site residues to a active configuration occurs upon substrate binding. The N-terminal peptide of ClpP-CMK is stabilized in a beta-hairpin conformation with the proximal N-terminal residues lining the axial channel and the loop extending beyond the apical surface of the heptameric ring. The lack of major substrate-induced conformational changes suggests that changes in ClpP structure needed to facilitate substrate entry or product release must be limited to rigid body motions affecting subunit packing or contacts between ClpP rings.

Amino Acid Sequence↗

PubMed2006

The structure of the extracellular domain of triggering receptor expressed on myeloid cells like transcript-1 and evidence for a naturally occurring soluble fragment.

Triggering receptor expressed on myeloid cells like transcript-1 (TLT-1) is an abundant platelet-specific, type I transmembrane receptor. The extracellular fragment of TLT-1 consists of a single, immunoglobulin-like domain connected to the platelet cell membrane by a linker region called the stalk. Here we present evidence that a soluble fragment of the TLT-1 extracellular domain is found in serum of humans and mice and that an isoform of similar mass is released from platelets following activation with thrombin. We also report the crystal structure of the immunoglobulin domain of TLT-1 determined at the resolution of 1.19 A. The structure of TLT-1 is similar to other immunoglobulin-like variable domains, particularly those of triggering receptor expressed on myeloid cells-1 (TREM-1), the natural killer cell-activating receptor NKp44, and the polymeric immunoglobulin receptor. Particularly interesting is a 17-amino acid segment of TLT-1, homologous to a fragment of murine TREM-1, which, in turn, showed activity in blocking the TREM-1-mediated inflammatory responses in mice. Structural similarity to TREM-1 and polymeric immunoglobulin receptor, and evidence for a naturally occurring soluble fragment of the TLT-1 extracellular domain, suggest that this immunoglobulin-like domain autonomously plays an as yet unidentified, functional role.

Animals↗

PubMed2004

Structure of the scorpion toxin BmBKTtx1 solved from single wavelength anomalous scattering of sulfur.

This report describes the crystal structure of the K(+) channel-blocking toxin, BmBKTx1, isolated recently from the venom of the scorpion Buthus martensi Karsch. This is only the second structure of the short-chain K(+) channel-blocking toxin from scorpion solved by means of X-ray crystallography. Additionally, reductive dimethylation of folded BmBKTx1 employed to induce its crystallization and solution of the structure based on the anomalous signal from the sulfur atoms make this example quite unique. The monomer of BmBKTx1 is formed by 31 amino acid residues, including 6 cysteines connected in 3 disulfide bridges. Crystals of this toxin belong to the space group P2(1) with two molecules present in the asymmetric unit. The unit cell parameters are a = 21.40 A, b=39.70 A, c=29.37 A, and beta-94.13 grades. Based on the high-quality dataset (anomalous signal) collected to the resolution 1.72A using the conventional X-radiation generator (lambda Cu, K alpha = 1.5478 A), the positions of sulfur atoms contributed by 12 cysteine residues have been identified, and subsequent improvement of the experimental phases have allowed structure solution. The final model was refined to the crystallographic R-factor of 0.166. The methyl groups on several lysine residues could be easily modeled into the electron density.

Amino Acid Sequence↗

PubMed2004

Sequence-altered peptide adopts optimum conformation for modification-dependent binding of the yeast tRNAPhe anticodon domain.

Amino acid contributions to protein recognition of naturally modified RNAs are not understood. Circular dichroism spectra and predictive software suggested that peptide tF2 (S1ISPW5GFSGL10 LRWSY15), selected from a phage display library to bind the modified anticodon domain of yeast tRNAPhe (ASL), adopted a beta-sheet structure. Ala residues incorporated at positions Pro4 and Gly6, both predicted to be involved in a turn, did not alter the peptide binding affinity for the ASLPhe, although major changes in the peptide's CD spectra were observed. Substitutions at three positions Pro4, Gly6, and Gly9, the latter not predicted to be in a turn, reduced the peptide's binding affinity to 4% of that of the unsubstituted tF2 and strongly influenced the peptide's secondary structure. The results suggest that peptides with different conformations, but similar affinities, adopt the optimal binding conformation, indicative of a structurally adaptive model of binding in which the modified RNA serves as a scaffold.

Anticodon↗

PubMed2003

Using capillary electrophoresis to study methylation effect on RNA-peptide interaction.

Methylation of RNA and proteins is one of a broad spectrum of post-transcriptional/translational mechanisms of gene expression regulation. Its functional signification is only beginning to be understood. A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the methylation effect on biomolecules interaction is presented. Two RNA-peptide systems were chosen for the study. The first one consists of a 17-nucleotide analogue (+27-+43) of the yeast tRNA(Phe) anticodon stem and loop domain (ASL(Phe)) containing three of the five naturally occurring modifications (2'-O-methylcytidine (Cm(32)), 2'-O-methylguanine (Gm(34)) and 5-methylcytidine (m(5)C(40))) (ASL(Phe)-Cm(32),Gm(34),m(5)C(40)) and a 15-amino-acid peptide (named t(F)2: Ser(1)-Ile-Ser-Pro-Trp(5)-Gly-Phe-Ser-Gly-Leu(10)-Leu- Arg-Trp-Ser-Tyr(15)) selected from a random phage display library (RPL). A peptide-concentration-dependent formation of an RNA-peptide complex was clearly observable by CEMSA. In the presence of the peptide the capillary electrophoresis (CE) peak for triply methylated ASL(Phe) shifted from 18.16 to 20.90 min. Formation of the complex was not observed when an unmethylated version of ASL(Phe) was used. The second system studied consisted of the (+18)-(+44) fragment of the trans-activation response element of human immunodeficiency virus type 1 (TAR RNA HIV-1) and a 9-amino-acid peptide of the trans-activator of transcription protein (Tat HIV-1) Tat(49-57)-NH(2) (named Tat1: Arg(49)-Lys-Lys-Arg(52)-Arg-Gln-Arg-Arg- Arg(57)-NH(2)). In the presence of Tat(49-57)-NH(2) a significant shift of migration time of TAR from 18.66 min to 20.12 min was observed. Methylation of a residue Arg(52)-->Arg(Me)(2), crucial for TAR binding, strongly disrupted formation of the complex. Only at a high micromolar peptide concentration a poorly shaped, broad peak of the complex was observed. CE was found to be an efficient and sensitive method for the analysis of methylation effects on interaction of biomolecules.

Amino Acid Sequence↗

PubMed2002

Structural requirements for conserved Arg52 residue for interaction of the human immunodeficiency virus type 1 trans-activation responsive element with trans-activator of transcription protein (49-57). Capillary electrophoresis mobility shift assay.

A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the interaction between the trans-activation response element (TAR) and the trans-activator of transcription protein (Tat) has been presented. The human immunodeficiency virus type 1 (HIV-1) Tat promotes elongation of viral mRNAs binding to the TAR. It has been suggested that a single, conserved arginine residue (presumably Arg52) within the arginine-rich region (ARR) of Tat plays the major role for the Tat-TAR recognition. To study structural requirements of the Arg52 position, Tat(49-57)-NH2 analogues substituted with nonencoded amino acids at the Arg52 position have been synthesized and their interaction with TAR has been studied by CEMSA. Using a linear polyacrylamide-coated capillary and a sieving polymer containing separation buffer, well separated and shaped peaks of free and bound TAR RNA were obtained. In the presence of Tat1 peptide bearing the native sequence of Tat(49-57) a significant shift of migration time of TAR from 18.66 min (RSD=1.4%) to 20.12 min (RSD=2.4%) was observed. We have found that almost every substitution within the guanidino group of the Arg52 [L-Arg52-->Cit, -->Orn, -->Arg(NO2), -->Arg(Me2)] strongly disrupted or abolished the TAR-Tat peptide interaction. Enantiomeric substitution, L-Arg52-->D-Arg was the only one which notably promoted TAR-Tat peptide interaction. The results demonstrate that the specific net of hydrogen bonds created by the guanidinio group of conserved Arg52 plays a crucial role for TAR-Tat HIV-1 recognition. The newly developed procedure describes for the first time use of CE to monitor RNA-peptide complex formation. The methodology presented should be generally applicable to study RNA-peptide (protein) interaction.

Amino Acid Sequence↗

PubMed2002

Interaction of RNA with phage display selected peptides analyzed by capillary electrophoresis mobility shift assay.

A sensitive capillary electrophoresis mobility shift assay (CEMSA) to analyze RNA/peptide interactions has been developed. Capillary electrophoresis (CE) has been adapted for investigating the interaction between variously methylated 17-nt analogs of the yeast tRNAPhe anticodon stem and loop domain (ASL(Phe)) and 15-amino-acid peptides selected from a random phage display library (RPL). A peptide-concentration-dependent formation of RNA/peptide complex was clearly visible during CEMSA. In the presence of peptide, the UV-monitored CE peak for ASLPhe with three of the five naturally occurring modifications (2'-O-methylcytidine (Cm32), 2'-O-methylguanine (Gm34) and 5-methylcytidine (m5C40) shifted from 18.16 to 20.90 min. The mobility shift was observed only for methylated RNA. The negative effects of diffusion, electroosmotic flow and adhesion of molecules to the capillary internal wall were suppressed by using a buffer containing a sieving polymer and a polyacrylamide-coated capillary. Under these conditions, well-shaped peaks and resolution of RNA free and bound to peptide were achieved. Peptide tF2, the most populated ligand in the RPL, specifically bound triply methylated ASLPhe in a methylated nucleoside-dependent manner. CE was found to be an efficient and sensitive method for the qualitative analysis of RNA-peptide interaction and should be generally applicable to the study of RNA-peptide (protein) interactions.

Amino Acid Sequence↗

PubMed2002

Mechanism of the contractile effects of galantide and Galanin(1-14) [alpha-aminobutyric acid]scyliorhinin-I in rat isolated fundus strips.

BACKGROUND: The study was undertaken to establish the pharmacological basis of the stimulatory activity of galantide (M15) and galanin(1-14)-(a-aminobutyric acid8-[Abu8])scyliorhinin-I [Scy-I] in gastric smooth muscle. MATERIAL/METHODS: Isotonic contractions of the isolated, longitudinal rat gastric fundus strips were recorded. RESULTS: Galanin, galanin(1-15)-NH2, M15 and galanin(1-14)-[Abu8]Scy-I elicited concentration-dependent contractions. Their EC50s equaled 12.95, 174, 70.06 and 187 nM respectively. Hill's coefficients for galanin and galanin(1-15)-NH2 were not different from unity, indicating an interaction of one peptide molecule with one receptor according to the principles of classical receptor theory. Hill's coefficients were 0.73 and 1.56 for M15 and galanin (1-14)-[Abu8]Scy-I, respectively, a value significantly different from unity. Cold-storage denervation, tetrodotoxin-TTX (1 mM), spantide (100 mM) and NK1-3 receptor antagonists SR140333, 48968, 142801 (up to 10 mM) notably diminished M15, galanin(1-14)-[Abu8]Scy-I, SP(5-11) and [Abu8]-Scy-I evoked contractions without affecting activities of galanin and galanin(1-15)-NH2. Additionally, cross-desensitization experiments attenuated activity of M15 and galanin(1-14)-[Abu8]Scy-I without any noticeable action on galanin or galanin(1-15)-NH2. CONCLUSIONS: The action of chimeric peptides on the smooth muscle of the rat gastrointestinal tract depended not only on the myogenic interaction of those peptides with galanin binding sites, but also on the activation of tachykinin receptors or the release of endogenous mediators from the presynaptic terminals.

Animals↗