Membrane Interactions of S100A12 (Calgranulin C)

Abstract

S100A12 (Calgranulin C) is a small acidic calcium-binding peripheral membrane protein with two EF-hand structural motifs. It is expressed in macrophages and lymphocytes and highly up-regulated in several human inflammatory diseases. In pigs, S100A12 is abundant in the cytosol of granulocytes, where it is believed to be involved in signal modulation of inflammatory process. In this study, we investigated the interaction of the porcine S100A12 with phospholipid bilayers and the effect that ions (Ca2+, Zn2+ or both together) have in modifying protein-lipid interactions. More specifically, we intended to address issues such as: (1) is the protein-membrane interaction modulated by the presence of ions? (2) is the protein overall structure affected by the presence of the ions and membrane models simultaneously? (3) what are the specific conformational changes taking place when ions and membranes are both present? (4) does the protein have any kind of molecular preferences for a specific lipid component? To provide insight into membrane interactions and answer those questions, synchrotron radiation circular dichroism spectroscopy, fluorescence spectroscopy, and surface plasmon resonance were used. The use of these combined techniques demonstrated that this protein was capable of interacting both with lipids and with ions in solution, and enabled examination of changes that occur at different levels of structure organization. The presence of both Ca2+ and Zn2+ ions modify the binding, conformation and thermal stability of the protein in the presence of lipids. Hence, these studies examining molecular interactions of porcine S100A12 in solution complement the previously determined crystal structure information on this family of proteins, enhancing our understanding of its dynamics of interaction with membranes.

Going deep into protein secondary structure with synchrotron radiation circular dichroism spectroscopy

“…The S100 proteins are members of this class and share a highly conserved helical folding across different species. The recombinant S100A12 (previously described in Garcia et al 2008) is a typical all-α protein that had its secondary structure first investigated with conventional CD and then with SRCD spectroscopy (Garcia et al (2013). Porcine S100A12 is a relatively small (11 kDa) calcium binding protein with two EFhand motifs and an additional zinc binding site at the C-terminus.…”

“…The major helix content estimated for S100A12 with CD spectroscopy (~75%) is in agreement with its classification in the CATH class. SRCD spectroscopy, however, was employed to monitor more accurately the secondary structure content of the recombinant protein (Garcia et al 2013) and to investigate the intermolecular interactions relevant to its role as a peripheral membrane protein. Such investigations require the use of biomembrane models (phospholipids vesicles) in high concentrations, which may promote light scattering that in turn hampers the quality of the CD spectrum in the CD method.…”

“…Estimations of the structural content from the S100A12 SRCD spectra revealed that the protein is mainly composed of α-helices (63%), but with a significant content of either turns or unordered structure (~22%). Therefore, a mismatching of 12% in the helix content was evidenced when estimations performed with conventional CD (Garcia et al 2008) were compared with those performed by SRCD (Garcia et al 2013). The authors of these studies attributed the mismatching to the limitations of the conventional CD method, especially in terms of monitoring the positive peak at 192 nm (which was completely observed with SRCD spectroscopy).…”

“…Many research groups have used SRCD spectroscopy to examine and estimate the secondary structure content of proteins (Lees et al 2006;Matsuo and Gekko 2013;Garcia et al 2013;Hussain et al 2016;Lopes et al 2016;Miler et al 2016). The technique has been successfully employed to characterize the insertion and orientation of helical peptides in lipid bilayers (Bürck et al 2016) or compare the behavior of globular and natively unfolded proteins (Ruskamo et al 2012;Yoneda et al 2017), investigate protein/peptide structure in model membranes (Miles et al 2008;Dreschler et al 2009), assess the dynamic and flexible conformation of unordered proteins (Lopes et al 2014a, b;Bremer et al 2017), evaluate protein:protein complex formation (Cowieson et al 2008;Lopes et al 2013), detect conformational changes induced by the binding to ligands (Lima et al 2013), perform fold recognition and obtain an accurate distinction between parallel and antiparallel β-sheets (Micsonai et al 2015) and identify the conformational changes associated with a mutant protein (Wallace et al 2004).…”

S100 calcium‐binding protein A12 as a diagnostic index for subclinical mastitis in cows

“…The mechanism for S100A12 role in the inflammation has not been clearly elucidated. S100A12 is mostly transported to the cell membrane or cytoskeleton from cytoplasm by its interaction with calcium (Garcia, Lopes, Costa-Filho, Wallace, & Araujo, 2013). Furthermore, S100A12 is secreted to the extracellular space in an autocrine or paracrine manner during the immune response.…”