Journal of Molecular Biology
Volume 381, Issue 5, 19 September 2008, Pages 1157-1167
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An Allosteric Circuit in Caspase-1

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Abstract

Structural studies of caspase-1 reveal that the dimeric thiol protease can exist in two states: in an on-state, when the active site is occupied, or in an off-state, when the active site is empty or when the enzyme is bound by a synthetic allosteric ligand at the dimer interface ∼ 15 Å from the active site. A network of 21 hydrogen bonds from nine side chains connecting the active and allosteric sites change partners when going between the on-state and the off-state. Alanine-scanning mutagenesis of these nine side chains shows that only two of them—Arg286 and Glu390, which form a salt bridge—have major effects, causing 100- to 200-fold reductions in catalytic efficiency (kcat/Km). Two neighbors, Ser332 and Ser339, have minor effects, causing 4- to 7-fold reductions. A more detailed mutational analysis reveals that the enzyme is especially sensitive to substitutions of the salt bridge: even a homologous R286K substitution causes a 150-fold reduction in kcat/Km. X-ray crystal structures of these variants suggest the importance of both the salt bridge interaction and the coordination of solvent water molecules near the allosteric binding pocket. Thus, only a small subset of side chains from the larger hydrogen bonding network is critical for activity. These form a contiguous set of interactions that run from one active site through the allosteric site at the dimer interface and onto the second active site. This subset constitutes a functional allosteric circuit or “hot wire” that promotes site-to-site coupling.

Introduction

Allostery, which refers to functional coupling between sites on proteins, is central to biological regulation. This phenomenon has been classically examined in hemoglobin and aspartate transcarbamoylase (ATCase), and has been generalized to many other regulatory enzymes involved in metabolic and signaling pathways, as well as in membrane and nuclear receptors (for a recent review, see Changeux and Edelstein1). While structural changes brought on by allostery are well-documented, the functional roles of amino acid side chains that couple conformational changes are less well understood. In this study, we begin to systematically examine the functional importance of amino acid side chains in coupling conformational changes between sites in caspase-1.

Caspases are dimeric thiol proteases that drive cellular processes such as apoptosis and inflammation. These enzymes have at least two conformational states that are observed crystallographically and represent both active and inactive zymogen conformations (for reviews, see Fuentes-Prior and Salvesen2 and Shi3). The active conformation of caspase-1 has been crystallized with substrate mimics bound to the active site, and these represent the on-state conformation of the enzyme.4, 5 Recently, specific allosteric small-molecule ligands have been captured at a cysteine residue at the dimer interface of apoptotic caspases-3 and -7,6 as well as the inflammatory caspase-1.4 These ligands bind to a site ∼ 15 Å from the active site and trap an inactive conformation of caspases. This off-state conformation is similar to the inactive zymogen form of caspase-76 or the ligand-free form of caspase-1,5 and therefore represents a natural state of these proteases (Fig. 1).

Caspase-1 shows positive cooperativity, indicating that binding of substrate at one active site enhances catalysis at the second site.4 Inspection of the X-ray structures for the active and allosterically inhibited enzyme suggests that coupling may be mediated by a network of 21 hydrogen bonding (H-bonding) interactions primarily involving nine side chains (Fig. 2). This network runs from one active site through the allosteric site to the second active site. Eight of the nine H-bonding residues in the network are completely conserved among inflammatory caspases-1 and -5, and a close homolog caspase-4. (Asp336 in caspase-1 is a histidine in caspases-4 and -5.) Overall, procaspase-1 has about a 45% sequence identity with procaspases-4 and -5, which have a 66% sequence identity with each other. One key interaction in caspase-1 appears to be a conserved salt bridge between Arg286 and Glu390, as the allosteric inhibitors in caspase-1 directly disrupt this network by preventing the salt bridge from forming.4

To better understand the importance of residues in the H-bonding network in caspase-1 for enzyme activity, we employed alanine-scanning mutagenesis. This approach has been proven effective for the systematic identification of functional “hot spots” in protein interfaces7 (for a recent review, see Moreira et al.8). Here, alanine-scanning mutational analysis shows that only four of the nine side chains, including the salt bridge, are significantly important for enzyme activity. These form a contiguous circuit of residues, or “hot wire,” that runs from one active site to the dimer interface and onto the allosteric site. Such allosteric circuits may be revealed by mutational analysis in other cooperative enzymes and help to identify important functional determinants for allosteric coupling and activity.

Section snippets

Mutational analysis of the H-bonding network in caspase-1

Active forms of caspase-1 have been crystallized while bound to the active-site inhibitors Ac-WEHD-CHO9 and z-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK)4 [Fig. 1, top; Protein Data Bank (PDB) ID code 2HQB]. They have also been crystallized as active-site ligand-free enzyme5 (Fig. 1, middle; PDB ID code 1SC1) and in the allosterically inhibited form4 (Fig. 1, bottom; PDB ID code 2FQQ). The active-site ligand-free and allosterically inhibited conformations are nearly identical, suggesting that

Discussion

These mutational and structural studies begin to reveal the critical features of an extensive and conserved H-bonding network that couples the functional sites in caspase-1. The alanine-scanning experiments indicate that only four of the nine H-bonding side chains have a significant effect on activity, especially the Arg286-Glu390 interaction. These form a contiguous chain of interactions that connects the active and allosteric sites in the protease.

It is remarkable that so many of the

Caspase-1 expression and purification

Recombinant caspase-1 was prepared by expression in E. coli as insoluble inclusion body, followed by refolding.5, 27 The p20 (residues 120–297) and p10 (residues 317–404) subunits of wild-type human caspase-1 were cloned into NdeI and EcoRI restriction endonuclease sites of the pRSET plasmid (Invitrogen, Carlsbad, CA). Site-directed mutagenesis was performed using the QuikChange Site-Directed Mutagenesis Kit from Stratagene (La Jolla, CA).

Caspase-1 subunits were expressed separately in E. coli

Acknowledgements

We wish to thank our colleagues in the laboratory for useful interactions, and Sunesis Pharmaceuticals for providing support for crystallography. This work was supported by the National Institutes of Health (grant ROI-AI070292 to J.A.W., grant F32AR052602 to J.M.S., and grant GMO7618 to D.D.).

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    Present address: Department of Protein Chemistry, Genentech, Inc., South San Francisco, CA 94080, USA.

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