Resonance assignments of the human AKAP13-PH domain and stabilizing DH helix

Abstract

The human AKAP13 protein contains DH and PH domains, which are responsible for its cell transforming activity. Despite its biomedical importance, the contribution of the PH domain to AKAP13 activity remains unclear and no three dimensional structure is available to date. Here we report the backbone and side chain (1)H, (13)C and (15)N resonance assignments of a 20 kDa construct comprising the uniformly (13)C and( 15)N labeled AKAP13-PH domain and an associated helix from the DH domain which is required for its stable expression. Resonance assignment has been achieved using conventional triple resonance experiments; 95% of all back bone resonances and more than 90% of side chain resonances have been successfully assigned. The (1)H, (13)C and (15)N chemical shifts have been deposited in BMRB with accession number of 16195.

Structural Insights into the Activation of the RhoA GTPase by the Lymphoid Blast Crisis (Lbc) Oncoprotein

“…The production of the AKAP-Lbc construct encompassing residues 2164–2346 (“DHαPH”) was as described previously (23). Expression was induced overnight by addition of 1 m m isopropyl 1-thio-β- d -galactopyranoside at 18 °C.…”

“…The structure of the DHαPH domain (500 μ m ) of onco-Lbc was determined using NMR spectra acquired at 297 K on Varian Inova 800- and 900-MHz spectrometers equipped with triple resonance cold probes with enhanced 13 C and 1 H sensitivity and z axis gradients using assigned 1 H, 13 C, and 15 N resonances (23). The protein samples were dissolved in H 2 O or 10% D 2 O and used for the acquisition of 13 C- and 15 N-resolved NOESY-HSQC experiments to estimate interproton distances from cross-peak volumes based on mixing times of 100 ms.…”