Inhibition of human leucocyte elastase by ursolic acid. Evidence for a binding site for pentacyclic triterpenes

Several pentacyclic triterpenoid metabolites of plant origin are inhibitors of hydrolysis of both synthetic peptide substrates and elastin by human leucocyte elastase (HLE). Ursolic acid, the most potent of these compounds, has an inhibition constant of 4-6 microM for hydrolysis of peptide substrates in phosphate-buffered saline. With tripeptide and tetrapeptide substrates, the inhibition is purely competitive, whereas with a shorter dipeptide substrate the inhibition is non-competitive, suggesting that ursolic acid interacts with subsite S3 of the extended substrate-binding domain in HLE, but not with subsites S1 and S2. The carboxy group at position 28 in the pentacyclic-ring system of the triterpenes contributes to binding to HLE, since replacement of this group with a hydroxy group, as in uvaol, the alcohol analogue of ursolic acid, reduces the potency of inhibition. The inhibitory potency of ursolic acid is also reduced by addition of 1 M-NaCl, further supporting a postulated electrostatic interaction between the negative charge on the triterpene and a positively charged residue on the enzyme, which we assign to the side chain of Arg-217, located in the vicinity of subsites S4 and S5 in HLE. These observations are consistent with a binding site for ursolic acid which extends from S3 towards S4 and S5 on the enzyme. Other triterpenes, including oleanolic acid, erythrodiol, hederagenin and 18 beta-glycyrrhetic acid, can also interact with this binding site. On the basis of these results we conclude that the extended substrate-binding domain of HLE can accommodate a variety of hydrophobic ligands, including not only such molecules as fatty acids [Ashe & Zimmerman (1977) Biochem. Biophys. Res. Commun. 75, 194-199; Cook & Ternai (1988) Biol. Chem. Hoppe-Seyler 369, 629-637], but also polycyclic molecules such as the pentacyclic triterpenoids.

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Insight into the mechanism of H+-coupled nucleobase transport

10.1101/2021.12.20.473561 ◽

2021 ◽

Author(s):

Jun Weng ◽

Xiaoming Zhou ◽

Pattama Wiriyasermkul ◽

Zhenning Ren ◽

Xiuwen Yan ◽

...

Keyword(s):

Ascorbic Acid ◽

Binding Site ◽

Substrate Binding ◽

Critical Role ◽

Binding Domain ◽

Purine Base ◽

Substrate Binding Site ◽

Functional Studies ◽

Dependent Transport ◽

Substrate Binding Domain

Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here we report the structure and function PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 Å resolution by X-ray crystallography. PurTCp forms a homodimer and each protomer has 14 transmembrane segments folded into a substrate-binding domain (core domain) and an interface domain (gate domain) A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines, and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.

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Substrate binding domain of murine leukemia virus reverse transcriptase. Identification of lysine 103 and lysine 421 as binding site residues.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)77926-5 ◽

1988 ◽

Vol 263 (4) ◽

pp. 1648-1653

Author(s):

A Basu ◽

V B Nanduri ◽

G F Gerard ◽

M J Modak

Keyword(s):

Reverse Transcriptase ◽

Binding Site ◽

Murine Leukemia Virus ◽

Substrate Binding ◽

Leukemia Virus ◽

Binding Domain ◽

Substrate Binding Domain ◽

Murine Leukemia

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Substrate-binding domain conformational dynamics mediate Hsp70 allostery

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1506692112 ◽

2015 ◽

Vol 112 (22) ◽

pp. E2865-E2873 ◽

Cited By ~ 67

Author(s):

Anastasia Zhuravleva ◽

Lila M. Gierasch

Keyword(s):

Binding Site ◽

Substrate Binding ◽

Conformational Dynamics ◽

Binding Domain ◽

Substrate Binding Site ◽

Functional Sites ◽

Binding Interface ◽

Wide Range ◽

Binding Cleft ◽

Substrate Binding Domain

Binding of ATP to the N-terminal nucleotide-binding domain (NBD) of heat shock protein 70 (Hsp70) molecular chaperones reduces the affinity of their C-terminal substrate-binding domain (SBD) for unfolded protein substrates. ATP binding to the NBD leads to docking between NBD and βSBD and releasing of the α-helical lid that covers the substrate-binding cleft in the SBD. However, these structural changes alone do not fully account for the allosteric mechanism of modulation of substrate affinity and binding kinetics. Through a multipronged study of the Escherichia coli Hsp70 DnaK, we found that changes in conformational dynamics within the βSBD play a central role in interdomain allosteric communication in the Hsp70 DnaK. ATP-mediated NBD conformational changes favor formation of NBD contacts with lynchpin sites on the βSBD and force disengagement of SBD strand β8 from strand β7, which leads to repacking of a βSBD hydrophobic cluster and disruption of the hydrophobic arch over the substrate-binding cleft. In turn, these structural rearrangements drastically enhance conformational dynamics throughout the entire βSBD and particularly around the substrate-binding site. This negative, entropically driven allostery between two functional sites of the βSBD–the NBD binding interface and the substrate-binding site–confers upon the SBD the plasticity needed to bind to a wide range of chaperone clients without compromising precise control of thermodynamics and kinetics of chaperone–client interactions.

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Defining the structure of the substrate-free state of the DnaK molecular chaperone

Biochemical Society Symposium ◽

10.1042/bss0680069 ◽

2001 ◽

Vol 68 ◽

pp. 69-82 ◽

Cited By ~ 3

Author(s):

Joanna F. Swain ◽

Renuka Sivendran ◽

Lila M. Gierasch

Keyword(s):

Binding Site ◽

Substrate Binding ◽

Binding Domain ◽

Full Length ◽

Free State ◽

Substrate Binding Site ◽

Terminal Domain ◽

C Terminus ◽

Ph Dependent ◽

Substrate Binding Domain

Members of the Hsp70 (heat-shock protein of 70 kDa) family of molecular chaperones bind to exposed hydrophobic stretches on substrate proteins in order to dissociate molecular complexes and prevent aggregation in the cell. Substrate affinity for the C-terminal domain of the Hsp70 is regulated by ATP binding to the N-terminal domain utilizing an allosteric mechanism. Our multi-dimensional NMR studies of a substrate-binding domain fragment (amino acids 387-552) from an Escherichia coli Hsp70, DnaK(387-552), have uncovered a pH-dependent conformational change, which we propose to be relevant for the full-length protein also. At pH 7, the C-terminus of DnaK(387-552) mimics substrate by binding to its own substrate-binding site, as has been observed previously for truncated Hsp70 constructs. At pH 5, the C-terminus is released from the binding site, such that DnaK is in the substrate-free state 10-20% of the time. We propose that the mechanism for the release of the tail is a loss of affinity for substrate at low pH. The pH-dependent fluorescence changes at a tryptophan residue near the substrate-binding pocket in full-length DnaK lead us to extend these conclusions to the full-length DnaK as well. In the context of the DnaK substrate-binding domain fragment, the release of the C-terminus from the substrate-binding site provides our first glimpse of the empty conformation of an Hsp70 substrate-binding domain containing a portion of the helical subdomain.

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