Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein

The ~24-amino-acid leucine-rich tandem repeat motif (PXXXXXLXXLXXLXLSXNXLXGXI) of carrot antifreeze protein comprises most of the processed protein and should contribute at least partly to the ice-binding site. Structural predictions using publicly available online sources indicated that the theoretical three-dimensional model of this plant protein includes a 10-loop β-helix containing the ~24-amino-acid tandem repeat. This theoretical model indicated that conservative asparagine residues create putative ice-binding sites with surface complementarity to the 1010 prism plane of ice. We used site-specific mutagenesis to test the importance of these residues, and observed a distinct loss of thermal hysteresis activity when conservative asparagines were replaced with valine or glutamine, whereas a large increase in thermal hysteresis was observed when phenylalanine or threonine residues were replaced with asparagine, putatively resulting in the formation of an ice-binding site. These results confirmed that the ice-binding site of carrot antifreeze protein consists of conservative asparagine residues in each β-loop. We also found that its thermal hysteresis activity is directly correlated with the length of its asparagine-rich binding site, and hence with the size of its ice-binding face.

Download Full-text

Calcium-Binding Generates the Semi-Clathrate Waters on a Type II Antifreeze Protein to Adsorb onto an Ice Crystal Surface

Biomolecules ◽

10.3390/biom9050162 ◽

2019 ◽

Vol 9 (5) ◽

pp. 162 ◽

Cited By ~ 5

Author(s):

Tatsuya Arai ◽

Yoshiyuki Nishimiya ◽

Yasushi Ohyama ◽

Hidemasa Kondo ◽

Sakae Tsuda

Keyword(s):

Binding Site ◽

Calcium Binding ◽

Crystal Surface ◽

Hydration Shell ◽

Antifreeze Protein ◽

Bound State ◽

Ice Crystal ◽

Prism Plane ◽

Ice Binding ◽

Significant Difference

Hydration is crucial for a function and a ligand recognition of a protein. The hydration shell constructed on an antifreeze protein (AFP) contains many organized waters, through which AFP is thought to bind to specific ice crystal planes. For a Ca2+-dependent species of AFP, however, it has not been clarified how 1 mol of Ca2+-binding is related with the hydration and the ice-binding ability. Here we determined the X-ray crystal structure of a Ca2+-dependent AFP (jsAFP) from Japanese smelt, Hypomesus nipponensis, in both Ca2+-bound and -free states. Their overall structures were closely similar (Root mean square deviation (RMSD) of Cα = 0.31 Å), while they exhibited a significant difference around their Ca2+-binding site. Firstly, the side-chains of four of the five Ca2+-binding residues (Q92, D94 E99, D113, and D114) were oriented to be suitable for ice binding only in the Ca2+-bound state. Second, a Ca2+-binding loop consisting of a segment D94–E99 becomes less flexible by the Ca2+-binding. Third, the Ca2+-binding induces a generation of ice-like clathrate waters around the Ca2+-binding site, which show a perfect position-match to the waters constructing the first prism plane of a single ice crystal. These results suggest that generation of ice-like clathrate waters induced by Ca2+-binding enables the ice-binding of this protein.

Download Full-text

Identification of a Putative Binding Site for Negatively Charged Surfaces in the Fibronectin Type II Domain of Human Factor XII

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614171 ◽

2000 ◽

Vol 84 (12) ◽

pp. 1057-1065 ◽

Cited By ~ 21

Author(s):

Henk te Velthuis ◽

Manuela Helmer-Citterich ◽

C. Hack ◽

Franca Citarella

Keyword(s):

Amino Acid ◽

Binding Site ◽

Three Dimensional ◽

Coagulation Factor ◽

Factor Xii ◽

Type Ii ◽

Three Dimensional Model ◽

Functional Sites ◽

Charged Surfaces ◽

Fibronectin Type

SummaryMonoclonal antibodies directed against functional sites of proteins provide useful tools for structure-function studies. Here we describe a mAb, KOK5, directed against the heavy chain region of human coagulation factor XII (FXII), which inhibits kaolin-induced clotting activity by preventing the binding of FXII to kaolin. Furthermore, mAb KOK5 enhances FXII susceptibility for cleavage by kallikrein and supports FXII autoactivation. Hence, mAb KOK5 likely is directed against the binding site of FXII for negatively charged surfaces. Screening of two phage-displayed random peptide libraries with mAb KOK5 selected phages that could be grouped on the basis of two amino acid consensus sequences: A) FXFQTPXW and B) HQ/LCTHR/KKC. Sequence A contains two motifs: one shares homology with FXII amino acid residues 30-33 (FPFQ), the second one with residues 57-60 (TPNF); both amino acid stretches belonging to the fibronectin type II domain of FXII. Sequence B also reveals homology with part of the fibronectin type II domain, i.e. the stretch 40-47 (HKCTHKGR). A three-dimensional model of FXII residues 28-65, obtained by homology modeling, indicated that the three amino acid stretches 30-33, 40-47 and 57-60 are close to each other and accessible for the solvent, i.e. in a form available for interaction with the monoclonal antibody, suggesting that mAb KOK5 recognizes a discontinuous epitope on the fibronectin type II domain of FXII. Peptides corresponding to FXII sequences 29-37 (FXII29-37) or 39-47 (FXII39-47), were synthesized and tested for the capability to inhibit FXII binding to negatively charged surfaces. Peptide FXII39-47 inhibited the binding of labeled FXII to kaolin and effectively prevented both dextran sulfate-and kaolin-induced activation of the contact system in plasma. Hence, we suggest that the fibronectin type II domain of FXII, in particular residues 39 to 47, contribute to the binding site of FXII for negatively charged surfaces.

Download Full-text

Cloning, post-translational modifications, heterologous expression and ligand-binding of boar salivary lipocalin

Biochemical Journal ◽

10.1042/bj3500369 ◽

2000 ◽

Vol 350 (2) ◽

pp. 369-379 ◽

Cited By ~ 22

Author(s):

Dietrich LOEBEL ◽

Andrea SCALONI ◽

Sara PAOLINI ◽

Carlo FINI ◽

Lino FERRARA ◽

...

Keyword(s):

Amino Acid ◽

Sequence Similarity ◽

Three Dimensional ◽

Protein Isoforms ◽

Cysteine Residues ◽

Protein Chemistry ◽

Single Individual ◽

Amino Acid Residues ◽

Three Dimensional Model ◽

Submaxillary Glands

Boar submaxillary glands produce the sex-specific salivary lipocalin (SAL), which binds steroidal sex pheromones as endogenous ligands. The cDNA encoding SAL was cloned and sequenced. From a single individual, two protein isoforms, differing in three amino acid residues, were purified and structurally characterized by a combined Edman degradation/MS approach. These experiments ascertained that the mature polypeptide is composed of 168 amino acid residues, that one of the three putative glycosylation sites is post-translationally modified and the structure of the bound glycosidic moieties. Two of the cysteine residues are paired together in a disulphide bridge, whereas the remaining two occur as free thiols. SAL bears sequence similarity to other lipocalins; on this basis, a three-dimensional model of the protein has been built. A SAL isoform was expressed in Escherichiacoli in good yields. Protein chemistry and CD experiments verified that the recombinant product shows the same redox state at the cysteine residues and that the same conformation is observed as in the natural protein, thus suggesting similar folding. Binding experiments on natural and recombinant SAL were performed with the fluorescent probe 1-aminoanthracene, which was efficiently displaced by the steroidal sex pheromone, as well as by several odorants.

Download Full-text

Amino acids important in enzyme activity and dimer stability for Drosophila alcohol dehydrogenase

Biochemical Journal ◽

10.1042/bj3080419 ◽

1995 ◽

Vol 308 (2) ◽

pp. 419-423 ◽

Cited By ~ 12

Author(s):

S W Chenevert ◽

N G Fossett ◽

S H Chang ◽

I Tsigelny ◽

M E Baker ◽

...

Keyword(s):

Enzyme Activity ◽

Alcohol Dehydrogenase ◽

Binding Site ◽

Aspartic Acid ◽

Three Dimensional ◽

Side Chain ◽

Wild Type ◽

Dimensional Model ◽

Three Dimensional Model ◽

Substrate Binding Site

We have determined the nucleotide sequences of eight ethyl methanesulphonate-induced mutants in Drosophila alcohol dehydrogenase (ADH), of which six were previously characterized by Hollocher and Place [(1988) Genetics 116, 253-263 and 265-274]. Four of these ADH mutants contain a single amino acid change: glycine-17 to arginine, glycine-93 to glutamic acid, alanine-159 to threonine, and glycine-184 to aspartic acid. Although these mutants are inactive, three mutants (Gly17Arg, Gly93Glu and Gly184Asp) form stable homodimers, as well as heterodimers with wild-type ADH, in which the wild-type ADH subunit retains full enzyme activity [Hollocher and Place (1988) Genetics 116, 265-274]. Interestingly, the Ala159Thr mutant does not form either stable homodimers or heterodimers with wild-type ADH, suggesting that alanine-159 is important in stabilizing ADH dimers. The mutations were analysed in terms of a three-dimensional model of ADH using bacterial 20 beta-hydroxysteroid dehydrogenase and rat dihydropteridine reductase as templates. The model indicates that mutations in glycine-17 and glycine-93 affect the binding of NAD+. It also shows that alanine-159 is part of a hydrophobic anchor on the dimer interface of ADH. Replacement of alanine-159 with threonine, which has a larger side chain and can hydrogen bond with water, is likely to reduce the strength of the hydrophobic interaction. The three-dimensional model shows that glycine-184 is close to the substrate binding site. Replacement of glycine-184 with aspartic acid is likely to alter the position of threonine-186, which we propose hydrogen bonds to the carboxamide moiety of NAD+. Also, the negative charge on the aspartic acid side chain may interact with the substrate and/or residues in the substrate binding site. These mutations provide information about ADH catalysis and the stability of dimers, which may also be useful in understanding homologous dehydrogenases, which include the human 17 beta-hydroxysteroid, 11 beta-hydroxysteroid and 15-hydroxyprostaglandin dehydrogenases.

Download Full-text