Hydrophobic sequences can substitute for the wild‐type N‐terminal sequence of cystatin A (stefin A) in tight binding to cysteine proteinases

Ylinenjärvi, K; Widersten, Mikael; Björk, Ingemar

doi:10.1046/j.1432-1327.1999.00312.x

Cited by 7 publications

(6 citation statements)

References 37 publications

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“…The RLV segment thus apparently interacts with papain in a similar manner as the authentic MIP segment of cystatin A, contributing ∼40% of the total unitary free energy of binding by maintaining a low k diss (15). This conclusion is in agreement with results of a previous study by random mutagenesis of the first four N-terminal residues of cystatin A and phage-display selection of high-affinity variants, showing that several, mainly hydrophobic, sequences can substitute for the MIP fragment of cystatin A in tight binding to papain (40). The lack of an effect of introducing the entire cystatin C N-terminal region into cystatin A on papain binding further indicates that residues N-terminal of the RLV segment do not enhance the interaction with this enzyme.…”

Section: Discussionsupporting

confidence: 91%

Grafting of Features of Cystatins C or B into the N-Terminal Region or Second Binding Loop of Cystatin A (Stefin A) Substantially Enhances Inhibition of Cysteine Proteinases

Pavlova

Björk

2003

Biochemistry

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Replacement of the three N-terminal residues preceding the conserved Gly of cystatin A by the corresponding 10-residue long segment of cystatin C increased the affinity of the inhibitor for the major lysosomal cysteine proteinase, cathepsin B, by approximately 15-fold. This tighter binding was predominantly due to a higher overall association rate constant. Characterization of the interaction with an inactive Cys29 to Ala variant of cathepsin B indicated that the higher rate constant was a result of an increased ability of the N-terminal region of the chimeric inhibitor to promote displacement of the cathepsin B occluding loop in the second binding step. The low dissociation rate constant for the binding of cystatin A to cathepsin B was retained by the chimeric inhibitor, which therefore had a higher affinity for this enzyme than any natural cystatin identified so far. In contrast, the N-terminal substitution negligibly affected the ability of cystatin A to inhibit papain. However, substitutions of Gly75 in the second binding loop of cystatin A by Trp or His, making the loop similar to those of cystatins C or B, respectively, increased the affinity for papain by approximately 10-fold. This enhanced affinity was due to both a higher association rate constant and a lower dissociation rate constant. Modeling of complexes between the two variants and papain indicated the possibility of favorable interactions being established between the substituting residues and the enzyme. The second-loop substitutions negligibly affected or moderately reduced the affinity for cathepsin B. Together, these results show that the inhibitory ability of cystatins can be substantially improved by protein engineering.

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Section: Discussionsupporting

confidence: 91%

Grafting of Features of Cystatins C or B into the N-Terminal Region or Second Binding Loop of Cystatin A (Stefin A) Substantially Enhances Inhibition of Cysteine Proteinases

Pavlova

Björk

2003

Biochemistry

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“…Members of the superfamily share regions of high sequence conservation, including the dipeptide GG (or GA) within the N‐terminal trunk, and the Q53‐G57 pentapeptide (conserved sequence QVVAG in stefin family) in the first binding loop. The importance of these regions for enzyme inhibition has been demonstrated1, 42 and confirmed by the tertiary structure of the complex between stefin B and papain. Deletion of residues at the N‐terminal trunk, where according to our study the order parameters in the monomer of stefin A are particularly low, results in a decreased affinity for papain and actinidin,43 while the K i value for cathepsin H of a mutant stefin B, where the N‐terminal MMC sequence was replaced by MRLV56, is increased by a factor of seven.…”

Section: Discussionmentioning

confidence: 84%

Comparison of backbone dynamics of monomeric and domain‐swapped stefin A

Japelj¹,

Waltho²,

Jerala³

2004

Proteins

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Three-dimensional domain swapping has been observed in increasing number of proteins and has been implicated in the initial stages of protein aggregation, including that of the cystatins. Stefin A folds as a monomer under native conditions, while under some denaturing conditions domain-swapped dimer is formed. We have determined the backbone dynamics of the monomeric and domain-swapped dimeric forms of stefin A by (15)N relaxation using a model-free approach. The overall correlation times of the molecules were determined to be 4.6 +/- 0.1 ns and 9.2 +/- 0.2 ns for the monomer and the dimer, respectively. In the monomer, decreased order parameters indicate an increased mobility for the N-terminal trunk, the first and the second binding loops. At the opposite side of the molecule, the loop connecting the alpha-helix with strand B, the beginning of strand B and the loop connecting strands C and D show increased localized mobility. In the domain-swapped dimer, a distinctive feature of the structure is the concatenation of strands B and C into a single long beta-strand. The newly formed linker region between strands B and C, which substitutes for the first binding loop in the monomer, has order parameters typical for the remainder of the beta-strands. Thus, the interaction between subunits that occurs on domain-swapping has consequences for the dynamics of the protein at long-range from the site of conformational change, where an increased rigidity in the newly formed linker region is accompanied by an increased mobility of loops remote from that site.

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“…In these studies the four N-terminal amino acids were randomly mutated and the resulting variants were expressed on the surface of the filamentous phage, and screened for binding to papain. Most of the variants that bound to the protease were found to have a glycine residue in position 4 (Ylinenjarvi et al 1999). Moreover, the glycine residue at position 10 of oryzacystatin-I was shown to be important for its inhibitory activity (Urwin et al 1995).…”

Section: Discussionmentioning

confidence: 99%

Comparative phylogenetic analysis of cystatin gene families from arabidopsis, rice and barley

Martínez¹,

Abraham²,

Carbonero³

et al. 2005

Mol Genet Genomics

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The plant cystatins or phytocystatins comprise a family of specific inhibitors of cysteine proteinases. Such inhibitors are thought to be involved in the regulation of several endogenous processes and in defence against pests and pathogens. Extensive searches in the complete rice and Arabidopsis genomes and in barley EST collections have allowed us to predict the presence of twelve different cystatin genes in rice, seven in Arabidopsis, and at least seven in barley. Structural comparisons based on alignments of all the protein sequences using the CLUSTALW program and searches for conserved motifs using the MEME program have revealed broad conservation of the main motifs characteristic of the plant cystatins. Phylogenetic analyses based on their deduced amino acid sequences have allowed us to identify groups of orthologous cystatins, and to establish homologies and define examples of gene duplications mainly among the rice and barley cystatin genes. Moreover, the absence of a counterpart between the two monocots, as well as strong variations in the motifs that interact with the cysteine proteinases, may be related to a species-specific evolutionary process. This cystatin classification should facilitate the assignment of proteinase specificities and functions to other cystatins as new information is obtained.

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Hydrophobic sequences can substitute for the wild‐type N‐terminal sequence of cystatin A (stefin A) in tight binding to cysteine proteinases

Cited by 7 publications

References 37 publications

Grafting of Features of Cystatins C or B into the N-Terminal Region or Second Binding Loop of Cystatin A (Stefin A) Substantially Enhances Inhibition of Cysteine Proteinases

Grafting of Features of Cystatins C or B into the N-Terminal Region or Second Binding Loop of Cystatin A (Stefin A) Substantially Enhances Inhibition of Cysteine Proteinases

Comparison of backbone dynamics of monomeric and domain‐swapped stefin A

Comparative phylogenetic analysis of cystatin gene families from arabidopsis, rice and barley

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