Conformational changes of calpain revealed by small angle X-ray scattering

INTRODUCTION: Calpains (EC 3.4.22.17) are a family of cytosolic calcium-dependent cysteine endopeptidases widely distributed in all mammalian cells and consisting of several genetically distinct isozymes structurally organized as heterodimers (1). The physiological role of calpains is related to the transduction of extra cellular signals mediated by changes in the permeability of membranes to Ca2+ or by the mobilization of this ion from internal stores. These proteins are also involved in other physiological and pathophysiological conditions such as cell cycle regulation, apoptosis, cytoskeletal remodeling, Alzheimer’s or Parkinson’s diseases and muscular dystrophies (2). The crystal structure of calcium-free recombinant human and rat m-calpain have recently been determined at 2.3 Å and 2.6 Å resolution respectively (3;4).Synchrotron radiation X-ray small angle scattering measurements were carried out on calpain isolated from human erythrocytes. Solutions of the proteinase were analyzed in the absence and in the presence of Ca 2+ and the structural effects of two different inhibitors, the synthetic inhibitor trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane (E64) and the neutral serine- and thiol-protease inhibitor leupeptin, were investigated.MATERIALS AND METHODS: Calpain was purified from erythrocytes according to Michetti et al. (5) and dissolved in Sodium Borate buffer pH 7.5 containing 0.1 mM EDTA in order to obtain a monodisperse protein solution. SAXS measurements were performed at the synchrotron radiation beam line D24 in the DCI storage ring of LURE (Laboratoire pour l’Utilisation du Rayonnement Electromagnetique, Orsay-Paris). The homogeneity of each sample was checked immediately before SAXS measurements by SE-HPLC chromatography (Waters 486 System with a Shodex Protein KW-802.5 column ). Eight frames of 100s each were recorded using a position sensitive proportional detector placed 1819 mm downstream from the sample so as to cover the range of momentum transfer Q from 0.01 to 0.15 Å-1 (Q = 4 sin/ where 2 is the scattering angle, * is the radiation wavelength, *=1.488 Å). The distance distribution function p(r), corresponding to the distribution of distances between any two volume elements within the protein particle, has been determined using the indirect transform method as implemented in the program GNOM. The scattering intensities were computed from the atomic coordinates of the crystal structure of the human m-calpain (1kfu.pdb) by using the program CRYSOL. The ab initio shape determination was performed with the dummy atom model (DAM) method (6) using the program DAMMIN running on a Silicon Graphics O2 workstation.RESULTS: The X-ray scattering model of the unligated enzyme in solution does not display any significant difference with that calculated from the crystal structure. The value of the radius of gyration calculated from the Guinier analysis of the scattering intensity results to be Rg = 35.8  0.4 Å. The molecular weight of the native protein calculated from the zero-angle intensity, calibrated by means of a reference sample has a value of 110  10 kDa, in close agreement with the values typical for both - and m- isoforms of calpains. Calculation of the p(r) function of the unliganded enzyme yields a value for the maximal diameter of 120 Å with a value of the radius of gyration of 36.3  0.4 Å, very close to that derived from the Guinier analysis. The SAXS pattern obtained for the native calpain in the presence of 100 M Ca2+ displays conspicuous differences with that of the unliganded protease indicating the formation of large aggregates in solution. In contrast, Ca2+ addition in the presence of the thiol proteinase inhibitor E64 or of the inhibitor leupeptin causes a small conformational change[...]