Deciphering the role of the electrostatic interactions involving Gly70 in eglin C by total chemical protein synthesis.


Abstract

Eglin c from the leech Hirudo medicinalis is a potent protein inhibitor of many serine proteinases including chymotrypsin and subtilisins. Unlike most small protein inhibitors whose solvent-exposed enzyme-binding loop is stabilized primarily by disulfide bridges flanking the reactive-site peptide bond, eglin c possesses an enzyme-binding loop supported predominantly by extensive electrostatic/H-bonding interactions involving three Arg residues (Arg48, Arg51, and Arg53) projecting from the scaffold of the inhibitor. As an adjacent residue, the C-terminal Gly70 participates in these interactions via its alpha-carboxyl group interacting with the side chain of Arg51 and the main chain of Arg48. In addition, the amide NH group of Gly70 donates an H-bond to the carbonyl C=O groups of Arg48 and Arg51. To understand the structural and functional relevance of the electrostatic/H-bonding network, we chemically synthesized wild-type eglin c and three analogues in which Gly70 was either deleted or replaced by glycine amide (NH(2)CH(2)CONH(2)) or by alpha-hydroxylacetamide (HOCH(2)CONH(2)). NMR analysis indicated that the core structure of eglin c was maintained in the analogues, but that the binding loop was significantly perturbed. It was found that deletion or replacement of Gly70 destabilized eglin c by an average of 2.7 kcal/mol or 20 degrees C in melting temperature. As a result, these inhibitors become substrates for their target enzymes. Binding assays on these analogues with a catalytically incompetent subtilisin BPN' mutant indicated that loss or weakening of the interactions involving the carboxylate of Gly70 caused a decrease in binding by approximately 2 orders of magnitude. Notably, for all four synthetic inhibitors, the relative free energy changes (DeltaDeltaG) associated with protein destabilization are strongly correlated (slope = 0.94, r(2) = 0. 9996) with the DeltaDeltaG values derived from a decreased binding to the enzyme. Study holds ProTherm entries: 7901 Extra Details: Truncated form of (1-70) S41C, eglinC enzyme-binding loop; H-bonding; free energy changes;,electrostatic interactions

Submission Details

ID: 7kfwLCpk

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:35 p.m.

Version: 1

Publication Details
Lu WY;Starovasnik MA;Dwyer JJ;Kossiakoff AA;Kent SB;Lu W,Biochemistry (2000) Deciphering the role of the electrostatic interactions involving Gly70 in eglin C by total chemical protein synthesis. PMID:10736156
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3TEC 1990-10-26T00:00:00+0000 2.0 CALCIUM BINDING TO THERMITASE. CRYSTALLOGRAPHIC STUDIES OF THERMITASE AT 0, 5 AND 100 MM CALCIUM
1TEC 1989-05-24T00:00:00+0000 2.2 CRYSTALLOGRAPHIC REFINEMENT BY INCORPORATION OF MOLECULAR DYNAMICS. THE THERMOSTABLE SERINE PROTEASE THERMITASE COMPLEXED WITH EGLIN-C
1SIB 1993-08-02T00:00:00+0000 2.4 REFINED CRYSTAL STRUCTURES OF SUBTILISIN NOVO IN COMPLEX WITH WILD-TYPE AND TWO MUTANT EGLINS. COMPARISON WITH OTHER SERINE PROTEINASE INHIBITOR COMPLEXES
1ACB 1991-11-08T00:00:00+0000 2.0 CRYSTAL AND MOLECULAR STRUCTURE OF THE BOVINE ALPHA-CHYMOTRYPSIN-EGLIN C COMPLEX AT 2.0 ANGSTROMS RESOLUTION
1EGL 1993-09-03T00:00:00+0000 0 THE SOLUTION STRUCTURE OF EGLIN C BASED ON MEASUREMENTS OF MANY NOES AND COUPLING CONSTANTS AND ITS COMPARISON WITH X-RAY STRUCTURES
2SEC 1988-09-05T00:00:00+0000 1.8 STRUCTURAL COMPARISON OF TWO SERINE PROTEINASE-PROTEIN INHIBITOR COMPLEXES. EGLIN-C-SUBTILISIN CARLSBERG AND CI-2-SUBTILISIN NOVO
1CSE 1988-06-03T00:00:00+0000 1.2 THE HIGH-RESOLUTION X-RAY CRYSTAL STRUCTURE OF THE COMPLEX FORMED BETWEEN SUBTILISIN CARLSBERG AND EGLIN C, AN ELASTASE INHIBITOR FROM THE LEECH HIRUDO MEDICINALIS. STRUCTURAL ANALYSIS, SUBTILISIN STRUCTURE AND INTERFACE GEOMETRY
4B1T 2012-07-12T00:00:00+0000 1.78 Structure of the factor Xa-like trypsin variant triple-Ala (TA) in complex with eglin C
4B2C 2012-07-13T00:00:00+0000 1.43 Structure of the factor Xa-like trypsin variant triple-Ala (TPA) in complex with eglin C
4B2B 2012-07-13T00:00:00+0000 1.36 Structure of the factor Xa-like trypsin variant triple-Ala (TGPA) in complex with eglin C

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 E Chymotrypsinogen A P00766 CTRA_BOVIN
100.0 I Chymotrypsinogen A P01051 ICIC_HIRME