FUNCTIONAL Ca2+INDUCED CONFORMATIONAL CHANGE IN HETERODIMERIC CALPAIN

INTRODUCTION: Calpains are a family of cytosolic cysteine proteinases which play a critical role in transducing extracellular signals involving changes in Ca2+ permeability of membranes or Ca2+ mobilization from internal stores. These proteases have been related to different indispensable physiological functions such as signal transduction, cell growth and differentiation, apoptosis, and they are involved in several pathological processes such as muscular dystrophies, cataract, Parkinson’s and Alzheimer’s diseases (1). Furthermore, there is emerging evidence that they play important roles as mediators of cell adhesion and motility in animal cells (2). Understanding the molecular mechanisms in the enzyme activation might help to unravel the calpain structures able to bind to the cellular membranes where this proteinase can exerts its activity. We have already described the overall structural modifications occurring in the native calpain in solution during its activation, as monitored by small angle X-ray scattering (SAXS) (3). Here we report a further investigation, aimed to clarify more into detail the calpain structures able to act at a cellular level.MATERIALS AND METHODS: Calpain was purified from erythrocytes as described elsewhere (4). SAXS experiments were carried out at the synchrotron radiation beam line D24 of LURE (Laboratoire pour l’Utilisation du Rayonnement Electromagnetique, Orsay-Paris). The radius of gyration (Rg) of the proteins in solution was determined on the basis of the Guinier approximation I(q) = I(0) exp(-Rg2 q2/3), where I(0) is the scattering intensity at zero angle. The maximum dimension of the particle (Dmax), the distance distribution function p(r)=1/2 2I(q) qr sin (qr) dq were evaluated using the program GNOM. The calculation of the theoretical SAXS pattern from the atomic coordinates was obtained with the program CRYSOL. The ab initio shape determination was performed with the dummy atom model (DAM) method (5) using the program DAMMIN running on a Silicon Graphics O2 workstation. Murine erythroleukaemia cells were analysed in the absence or in the presence of 1 M ionophore (A23187) and were pre-treated with the calpain inhibitor and 50 M Ca2+. The calpain localization was assessed by confocal laser microscopy (mod. Radiance 2000, Biorad) using the antibody Mab53 raised against erythrocyte calpain and an anti-mouse fluorescein conjugated.RESULTS: We show that the scattering pattern of the unliganded enzyme does not present any significant difference with that calculated from the atomic coordinates with a value of radius of gyration of 36.3  0.4 Å and a Dmax of 120 Å. Moreover, the addition of Ca2+ determines the formation of large soluble aggregates, indicating the exposure of hydrophobic patches on the surface of the proteinase. On the other hand, Ca2+ addition in the presence of the thiol proteinase inhibitor E64 or of the inhibitor leupeptin causes a small conformational change leading to a transition to an extended conformation with no dissociation of the heterodimer yielding a of radius of gyration of 38.5  0.7 Å and a Dmax of 130 Å. The comparison of the structural models obtained from the scattering pattern with the crystal structure indicates the existence of a conformational change localized at the level of the L-subunit, and seems to confirm the presence of mutual domain movement leading to an active conformation of calpain. We analysed the reversibility of the aggregation phenomenon observed at 100 M free Ca2+. We obtained similar SAXS patterns either removing Ca2+ by adding 1 mM EDTA or in the presence of chaotropic agents (i.e. 100 mM KSCN, NaCl or KCl). These SAXS curves were not superimposable to that of the Ca2+-free form of calpain, indicating a non-complete reversibility of [...]