Characterization of the structure and self-recognition of the human centrosomal protein NA14: implications for stability and function

Abstract

The protein NA14 is a key adaptor protein mediating the intermolecular interactions of microtubules and Spastin. To gain insight into its structure and function, we have expressed, purified and characterized human NA14 and some variants. NA14 is rather insoluble and tends to oligomerize and form fibrils. Successive mutation of the three Cys and two potentially exposed Leu residues (83 and 93) yielded a water-soluble quintuple variant, named 3CS-2LR. NA14 and its variants have a high helical content as determined by circular dichroism (CD). Based on nuclear magnetic resonance data of the quintuple mutant and the wild-type (wt) protein in the presence of dodecylphosphocholine micelles, the N-(M1-N13) and C-termini (K105-S119) were found to lack preferred structure. The remaining residues (14-104) participate in NA14 self-association, probably by forming a parallel coiled-coil structure. We hypothesize that Leu 83 and Leu 93 mediate interactions among NA14, Spastin and microtubules. We have also examined urea and thermal denaturation of the quintuple and other NA14 variants at different pH values by CD. The pH dependence of the conformational stability and the elevated native-state pK(a) determined for the two conserved Tyr allow us to propose that the NA14 structure may be stabilized by two Glu-COO(-) ||| HO-Tyr H-bonds, highly conserved in NA14-like proteins in other species.

Conformational characterization of the intrinsically disordered protein Chibby: Interplay between structural elements in target recognition

“…The mapping of coiled-coil domains using NMR spectroscopy has proven to be a challenge for not only Cby, but also for other partially unstructured proteins such as CHOP, Par-4, and NA14. [31][32][33] For those three proteins, the only observable HSQC peaks correspond to residues located in disordered regions. As demonstrated in this study, HDX-MS is a valuable alternative technique for deciphering which residues make up these coiled-coil domains.…”

“…The mapping of coiled‐coil domains using NMR spectroscopy has proven to be a challenge for not only Cby, but also for other partially unstructured proteins such as CHOP, Par‐4, and NA14 . For those three proteins, the only observable HSQC peaks correspond to residues located in disordered regions.…”

“…Our previous work showed that most of the NMR resonances for Cby's C‐terminal half are too broad to be observed under non‐denaturing conditions, likely due to the monomer/dimer conformational exchange or formation of higher oligomeric forms . This behavior is not unique to Cby, as severe NMR broadening has been seen in other proteins that are comprised of long disordered regions and coiled‐coil motifs …”

“…17 This behavior is not unique to Cby, as severe NMR broadening has been seen in other proteins that are comprised of long disordered regions and coiled-coil motifs. [31][32][33] Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is another widely used technique to analyze protein folding, structure, dynamics, and interactions. [34][35][36][37][38] Backbone amide hydrogens within disordered regions will exchange rapidly upon D 2 O exposure, in comparison to residues in well-folded, rigid regions, where slow D 2 O uptake is mediated by conformational fluctuations that lead to transient opening of hydrogen bonds.…”

Spastin-Interacting Protein NA14/SSNA1 Functions in Cytokinesis and Axon Development

“…In fact, previous studies have shown that spastin acts as a microtubule-severing protein in mammalian cells, and expression of spastin mutants unable to hydrolyze ATP result in the increased formation of stable bundles of microtubules [9]. Moreover, Rodríguez-Rodríguez et al [48] have shown that NA14 can create a dynamic matrix between microtubules and spastin, providing a scaffold for anchoring proteins that are involved in microtubule nucleation and axonal development. A transport role for NA14 also has precedent, since NA14 has previously been implicated in transport of the orphan receptor TPRA40/GPR175, and the interaction with NA14 is required for the effects of TPRA40/GPR175 on cell division in mouse embryos [49].…”