Wyniki wyszukiwania - Biblioteka Nauki

1

Structure-function relationship of serine protease-protein inhibitor interaction.

100%

Otlewski J. , Jaskólski M. , Buczek O. , Cierpicki T. , Czapińska H. , Krowarsch D. , Smalas A. O. , Stachowiak D. , Szpineta A. , Dadlez M.

Acta Biochimica Polonica

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2001

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tom 48

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nr 2

419-428

EN

We report our progress in understanding the structure-function relationship of the interaction between protein inhibitors and several serine proteases. Recently, we have determined high resolution solution structures of two inhibitors Apis mellifera chymotrypsin inhibitor-1 (AMCI-I) and Linum usitatissimum trypsin inhibitor (LUTI) in the free state and an ultra high resolution X-ray structure of BPTI. All three inhibitors, despite totally different scaffolds, contain a solvent exposed loop of similar conformation which is highly complementary to the enzyme active site. Isothermal calorimetry data show that the interaction between wild type BPTI and chymotrypsin is entropy driven and that the enthalpy component opposes complex formation. Our research is focused on extensive mutagenesis of the four positions from the protease binding loop of BPTI: P1, P1', P3, and P4. We mutated these residues to different amino acids and the variants were characterized by determination of the association constants, stability parameters and crystal structures of protease-inhibitor complexes. Accommodation of the P1 residue in the S1 pocket of four proteases: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 variants. High resolution X-ray structures of ten complexes between bovine trypsin and P1 variants of BPTI have been determined and compared with the cognate P1 Lys side chain. Mutations of the wild type Ala16 (P1') to larger side chains always caused a drop of the association constant. According to the crystal structure of the Leu16 BPTI-trypsin complex, introduction of the larger residue at the P1' position leads to steric conflicts in the vicinity of the mutation. Finally, mutations at the P4 site allowed an improvement of the association with several serine proteases involved in blood clotting. Conversely, introduction of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing effect on the complex with these proteases.

2

Signalling protein inhibitors via the combinatorial modification of peptide scaffolds

100%

Lawrence D. S.

Cellular and Molecular Biology Letters

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2005

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tom 10

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nr Suppl.2

3

Characterization of a novel protein inhibitor of protein kinases specific to acidic ribosomal proteins

100%

Pilecki M. , Szyszka R.

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tom 39

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nr 1

4

Protein inhibitors of serine proteinases

86%

Otlewski J. , Krowarsch D. , Apostoluk W.

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1999

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tom 46

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nr 3

EN

Serine proteinases and their natural protein inhibitors belong to the most intensively studied models of protein-protein recognition. Protein inhibitors do not form a single group but can be divided into about 20 different families. Global structures of proteins representing different inhibitor families are completely different and comprise α-helical proteins, β-sheet proteins,α/β-proteins and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished: canonical (standard mechanism) inhibitors, non-canonical inhibitors, and serpins. The canonical inhibitor binds to the enzyme through the exposed and convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is consistently very similar and resembles that of an ideal substrate. Non-canonical inhibitors, originating from blood sucking organisms, specifically block enzymes of the blood clotting cascade. The interaction is mediated through inhibitor N-terminus which binds to the proteinase forming a parallel β-sheet. There are also extensive secondary interactions which provide an additional buried area and contribute significantly to the strength and specificity of recognition. Serpins are major proteinase inhibitors occurring in plasma. Similarly to canonical inhibitors, serpins interact with their target proteinases in a substrate-like manner. However, in the case of serpins, cleavage of a single peptide bond in a flexible and exposed binding loop leads to dramatic structural changes.

5

Interaction of cysteine proteinase inhibitor with liposomes. II. Effect on the inhibition of cathepsin

72%

Gutowicz J. , Saleh J. , Pola A. , Siewinski M.

Cellular and Molecular Biology Letters

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1999

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tom 04

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nr 2

6

Structure-function relationship of serine protease-protein inhibitor interaction

72%

Otlewski J. , Jaskolski M. , Buczek O. , Cierpicki T. , Czapinska H. , Krowarsch D. , Smalas A. O. , Stachowiak D. , Szpineta A. , Dadlez M.

Acta Biochimica Polonica

|

2001

|

tom 48

|

nr 2

EN

We re port our prog ress in un der stand ing the struc ture-function re la tion ship of the interaction between protein inhibitors and several serine proteases. Recently, we have de ter mined high res o lu tion so lu tion struc tures of two in hib i tors Apis mellifera chymotrypsin in hib i tor-1 (AMCI-I) and Linum usitatissimumtrypsin in hib i tor (LUTI) in the free state and an ul tra high res o lu tion X-ray struc ture of BPTI. All three in hib i tors, de spite to tally dif fer ent scaf folds, con tain a sol vent ex posed loop of sim i lar con- for ma tion which is highly com ple men tary to the en zyme ac tive site. Iso ther mal calorim e try data show that the in ter ac tion be tween wild type BPTI and chymotrypsin is entropy driven and that the enthalpy com po nent op poses com plex for ma tion. Our research is fo cused on ex ten sive mu ta gen e sis of the four po si tions from the pro te ase bind ing loop of BPTI: P1, P1', P3, and P4. We mu tated these res i dues to dif fer ent amino ac ids and the vari ants were char ac ter ized by de ter mi na tion of the as so ci a tion con stants, sta bil ity pa ram e ters and crys tal struc tures of pro te ase–in hib i tor complexes. Ac com mo da tion of the P1 res i due in the S1 pocket of four pro teas es: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 vari ants. High res o lu tion X-ray struc tures of ten com plexes be tween bo vine trypsin and P1 vari ants of BPTI have been de ter mined and com pared with the cog nate P1 Lysside chain. Mu ta tions of the wild type Ala16 (P1') to larger side chains al ways caused a drop of the as so ci a tion con stant. Ac cord ing to the crys tal struc ture of the Leu16 BPTI–trypsin com plex, in tro duc tion of the larger res i due at the P1' po si tion leads to steric con flicts in the vi cin ity of the mu ta tion. Finally, mu ta tions at the P4 site al lowed an im prove ment of the as so ci a tion with sev eral serine pro teas es in volved in blood clot ting. Con versely, in tro duc tion of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing ef fect on the com plex with these pro teas es.