NMR characterisation of the minimal interacting regions of centrosomal proteins 4.1R and NuMA1: effect of phosphorylation

Abstract

BackgroundSome functions of 4.1R in non-erythroid cells are directly related with its distinct sub-cellular localisation during cell cycle phases. During mitosis, 4.1R is implicated in cell cycle progression and spindle pole formation, and co-localizes with NuMA1. However, during interphase 4.1R is located in the nucleus and only partially co-localizes with NuMA1.ResultsWe have characterized by NMR the structural features of the C-terminal domain of 4.1R and those of the minimal region (the last 64 residues) involved in the interaction with NuMA1. This subdomain behaves as an intrinsically unfolded protein containing a central region with helical tendency. The specific residues implicated in the interaction with NuMA1 have been mapped by NMR titrations and involve the N-terminal and central helical regions. The segment of NuMA1 that interacts with 4.1R is phosphorylated during mitosis. Interestingly, NMR data indicates that the phosphorylation of NuMA1 interacting peptide provokes a change in the interaction mechanism. In this case, the recognition occurs through the central helical region as well as through the C-terminal region of the subdomain meanwhile the N-terminal region do not interact.ConclusionsThese changes in the interaction derived from the phosphorylation state of NuMA1 suggest that phosphorylation can act as subtle mechanism of temporal and spatial regulation of the complex 4.1R-NuMA1 and therefore of the processes where both proteins play a role.

Structural insight into the XTACC3/XMAP215 interaction from CD and NMR studies on model peptides

“…In this regard, our CD data show that all peptides become more structured at lower pH values (at least in the presence of DPC micelles that mimics the hydrophobic and crowded cellular location) suggesting that they are more stable in the conditions near their physiological environment. Similar results were described for other centrosomal proteins …”

“…It was shown previously that peptides from many centrosomal proteins tend to aggregate . Thus, we analyzed the individual peptides over the concentrations range of 6–60 μM at pH 5.5 in H 2 O.…”

The Dynamic Multisite Interactions between Two Intrinsically Disordered Proteins

“…[6] More detailed biochemical characterizations of CTD showed that a1 5kDa fragment is stable under chymotryptic digestion and is most likely ag lobular folded domain. [8] On the other hand, the C-terminal region of NuMA, which contains binding sites for several proteins involved in the control of mitosis, [2a, 9] is also unstructured according to sequence analysis and previous structural studies. [8] On the other hand, the C-terminal region of NuMA, which contains binding sites for several proteins involved in the control of mitosis, [2a, 9] is also unstructured according to sequence analysis and previous structural studies.…”

“…[7] Recent NMR characterizations of 4.1R-CTD demonstrated that it is most likely intrinsically disordered or unstructured. [8] On the other hand, the C-terminal region of NuMA, which contains binding sites for several proteins involved in the control of mitosis, [2a, 9] is also unstructured according to sequence analysis and previous structural studies. [10] Ac on- ventional diagram of interactions between intrinsically disordered proteins (IDPs) involve adisorder-to-order transition to form as table structured complex.…”

The Dynamic Multisite Interactions between Two Intrinsically Disordered Proteins

“…More detailed biochemical characterizations of CTD showed that a 15 kDa fragment is stable under chymotryptic digestion and is most likely a globular folded domain . Recent NMR characterizations of 4.1R‐CTD demonstrated that it is most likely intrinsically disordered or unstructured . On the other hand, the C‐terminal region of NuMA, which contains binding sites for several proteins involved in the control of mitosis, is also unstructured according to sequence analysis and previous structural studies .…”