Human FEZ1 has characteristics of a natively unfolded protein and dimerizes in solution

Abstract

The fasciculation and elongation protein Zeta 1 (FEZ1) is the mammalian orthologue of the Caenorhabditis elegans protein UNC-76, which is necessary for axon growth. Human FEZ1 interacts with Protein Kinase C (PKC) and several regulatory proteins involved in functions ranging from microtubule associated transport to transcriptional regulation. Theoretical prediction, circular dichroism, fluorescence spectroscopy, and limited proteolysis of recombinant FEZ1 suggest that it contains disordered regions, especially in its N-terminal region, and that it may belong to the group of natively unfolded proteins. Small angle X-ray scattering experiments indicated a mainly disordered conformation, proved that FEZ1 is a dimer of elongated shape and provided overall dimensional parameters for the protein. In vitro pull down experiments confirmed these results and demonstrated that dimerization involves the N-terminus. Ab-initio 3D low resolution models of the full-length conformation of the dimeric constructs 6xHis-FEZ1(1-392) and 6xHis-FEZ1(1-227) were obtained. Furthermore, we performed in vitro phosphorylation assays of FEZ1 with PKC. The phosphorylation occurred mainly in its C-terminal region, and does not cause any significant conformational changes, but nonetheless inhibited its interaction with the FEZ1 interacting domain of the protein CLASP2 in vitro. The C terminus of FEZ1 has been reported to bind to several interacting proteins. This suggests that FEZ1 binding and transport function of interacting proteins may be subject to regulation by phosphorylation.

The SCHOOL of nature: II. Protein order, disorder, and oligomericity in transmembrane signaling

“…46 Specific complex formation between IDPs is quite unusual, but not unprecedented. 47 Homodimerization of IDPs was first reported in 2004 for a novel class of signaling-related IDPs 9 and later confirmed for other IDPs [48][49][50][51] extending the phenomenon to different classes of IDPs and suggesting physiological relevance. It should be noted that in most of these studies, dimerization is accompanied by a mutual or "synergistic" 47 folding of two IDP molecules at the interaction interface (Fig.…”

“…IDPs are often referred to as "remaining predominantly disordered" or "largely unfolded" upon dimerization or interaction with other proteins or lipids, 35,[39][40][41][42][43][44][45][48][49][50][51] meaning that the protein regions flanking the interaction interface but not the interface itself remain disordered. Recently, it has been suggested to term this mode of interaction "the flanking fuzziness" in contrast to "the random fuzziness" when the IDP remains entirely disordered in the bound state.…”

“…71,72,74 Later, other IDPs have also been found to form specific homodimers [48][49][50][51] and shown to function through dimer (e.g., NKG2D, CD94/NKG2C, KIR2DS, NKp30, NKp44 and NKp46), immunoglobulin (Ig)-like transcripts and leukocyte Ig-like receptors (ILTs and LIRs, respectively), signal regulatory proteins (SIRPs), dendritic cell immunoactivating receptor (DCAR), myeloid DNAX adapter protein of 12 kD (DAP12)-associating lectin 1 (MDL-1), blood DC antigen 2 protein (BDCA2), novel immune-type receptor (NITR), myeloid-associated Ig-like receptor (MAIR-II), triggering receptors expressed on myeloid cells (TREMs) and the platelet collagen receptor, glycoprotein VI (GPVI). For more information on the structure and function of these and other MIRRs, I refer the reader to recent reviews.…”

Biophysical characterization of the isolated C‐terminal region of the transient receptor potential vanilloid 1

“…This latter domain has been demonstrated to form homodimers without a disorder-order transition [48]. Interestingly enough, the T-cell receptor belongs to a group of receptors whose signaling depends on a two-step monomer-dimer-tetramer fast dynamic equilibrium [49]; this oligomerization of the intrinsically disordered region is essential to the receptor signaling [50,51]. We hypothesize that the C-TRPV1 self-associated species might be originated by suffering a very similar process, being the domain oligomerization the essential step for an effective signaling of the whole TRPV1.…”

Unusual biophysics of immune signaling-related intrinsically disordered proteins

“…Intriguingly, these studies not only challenge the generally accepted paradigms in protein functional role of IDP homointeractions in transmembrane signaling. 1,3,9,10 Later, in independent studies by several groups, other IDPs have also been found to form specific homodimers [11][12][13][14] and shown to function through dimer formation, 11,14 confirming a direct functional link between protein intrinsic disorder and oligomericity.…”