Membrane-Docking Loops of the cPLA2 C2 Domain:  Detailed Structural Analysis of the Protein−Membrane Interface via Site-Directed Spin-Labeling

Abstract

C2 domains are protein modules found in numerous eukaryotic signaling proteins, where their function is to target the protein to cell membranes in response to a Ca 2+ signal. Currently, the structure of the interface formed between the protein and the phospholipid bilayer is inaccessible to high-resolution structure determination, but EPR site-directed spin-labeling can provide a detailed medium-resolution view of this interface. To apply this approach to the C2 domain of cytosolic phospholipase A 2 (cPLA 2 ), single cysteines were introduced at all 27 positions in the three Ca 2+ -binding loops and labeled with a methanethiosulfonate spin-label. Altogether, 24 of the 27 spin-labeled domains retained Ca 2+ -activated phospholipid binding. EPR spectra of these 24 labeled domains obtained in the presence and absence of Ca 2+ indicate that Ca 2+ binding triggers subtle changes in the dynamics of two localized regions within the Ca 2+ -binding loops: one face of the loop 1 helix and the junction between loops 1 and 2. However, no significant changes in loop structure were detected upon Ca 2+ binding, nor upon Ca 2+ -triggered docking to membranes. EPR depth parameters measured in the membrane-docked state allow determination of the penetration depth of each residue with respect to the membrane surface. Analysis of these depth parameters, using an improved, generalizable geometric approach, provides the most accurate picture of penetration depth and angular orientation currently available for a membrane-docked peripheral protein. Finally, the observation that Ca 2+ binding does not trigger large rearrangements of the membrane-docking loops favors the electrostatic switch model for Ca 2+ activation and disfavors, or places strong constraints on, the conformational switch model. Cellular signals are transmitted by a variety of mechanisms, including the release of small molecule second messengers such as Ca 2+ or phosphoinositides, protein translocation between cellular compartments, and protein posttranslational modification. C2 domains are protein-signaling modules found in numerous signaling proteins (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Usually, the function of such C2 domains is to trigger the translocation of proteins to specific cellular membranes in response to a Ca 2+ signal. This targeting increases the likelihood of interaction of the signaling protein with its downstream target, often a membrane-bound lipid or protein.Cytosolic phospholipase A 2 (cPLA 2 ) 1 hydrolyzes lipids containing arachadonic acid and thereby releases this important precursor from nuclear and endoplasmic reticulum (ER) † Support provided by NIH Grant GM R01-63235 (to J.J.F.).© 2003 American Chemical Society * To whom correspondence should be addressed. falke@colorado.edu. Tel: (303) 492-3503. 1 Abbreviations: cPLA 2 , cytosolic phospholipase A 2 ; PS, phosphati-dylserine; PC, phosphatidylcholine; PE, phosphoethanolamine; MTSSL, 1-oxyl-2,2,5,5-tetramethyl-Δ 3 -pyrroline-3-methyl methanethiosulfonate; NiEDDA, Ni(II) ethylenediamine...

Ceramide-1-phosphate Binds Group IVA Cytosolic Phospholipase a2 via a Novel Site in the C2 Domain

“…Our earlier study demonstrated that the effects of C1P binding are more prominent on the isolated C2 domain than full-length cPLA 2 ␣, suggesting that C1P effects are more local to the C2 domain binding of cPLA 2 ␣. The current study opens an avenue to investigate the nature and orientation of cPLA 2 ␣ as well as its isolated C2 domain at the C1P-and PtdIns(4,5)P 2 -containing membrane interface through lipid penetration analysis (10), EPR (37,41,42), x-ray reflectivity studies (38), or molecular dynamics simulations (41).…”

Structure and Dynamics of a Helical Hairpin that Mediates Calcium-dependent Membrane Binding of Annexin B12

“…Indeed, R1 side chains on the surface of bacteriorhodopsin, facing the headgroups of the bilayer, have intermediate mobility (15) much higher than that observed for 144R1 on annexin B12 bound to bilayers. Relevant to the present work are SDSL studies of the Ca 2ϩ -dependent binding to phospholipid bilayers of the C2 domains of phospholipase A 2 (27) and protein kinase C (28) to phospholipid bilayers. In these studies, R1 was placed at sites within the Ca 2ϩ -binding loops of these proteins, and some of these sites localized to the headgroup region of the bilayer in the bound state.…”

Ca2+ Activation of the cPLA2 C2 Domain: Ordered Binding of Two Ca2+ Ions with Positive Cooperativity

“…The Ca 2+ dependence of the fluorescence increase was quantified by plotting the fluorescence change (ΔF) as a function of free Ca 2+ concentration ([Ca 2+ ]) and best-fitted using a Hill equation (18): (1) where ΔF max represents the calculated maximal fluorescence change (normalized to 1.0 to facilitate comparison of different mutant samples), H represents the Hill coefficient, and [Ca 2+ ] 1/2 represents the free Ca 2+ concentration that induces half-maximal fluorescence change. Best-fitting was carried out via nonlinear least-squares analysis in Kaleidagraph 3.5 (Synergy).…”