1H, 13C and 15N resonance assignments of human BASP1

Brain acid-soluble protein 1 (BASP1, CAP-23, NAP-22) appears to be implicated in diverse cellular processes. An N-terminally myristoylated form of BASP1 has been discovered to participate in the regulation of actin cytoskeleton dynamics in neurons, whereas non-myristoylated nuclear BASP1 acts as co-suppressor of the potent transcription regulator WT1 (Wilms’ Tumor suppressor protein 1). Here we report NMR chemical shift assignment of recombinant human BASP1 fused to an N-terminal cleavable His6-tag.

involved in neurite outgrowth and plasma membrane organization (Korshunova et al. 2008). It is able to interact with the inner leaflet of the plasma membrane via its myristoyl-anchor and sequesters Phosphatidyl-inositol-4,5diphosphate (PIP2) into lipid rafts (Epand et al. 2004;Shaw et al. 2006). Recently it has been shown that liposomes containing anionic phospholipids induce oligomerization of BASP1. Interaction with calmodulin is followed by dissociation of BASP1 from the membrane and disruption of the oligomers (Zakharov and Mosevitsky 2010). Additionally, BASP1 is under the control of protein kinase C (PKC), which phosphorylates BASP1 at Ser5. It is hypothesized that phosphorylation leads to the disruption of the interaction of the N-terminal positive effector domain of BASP1 with anionic phospholipids (Laux et al. 2000).
Furthermore, non-myristoylated BASP1 appears to influence transcription regulation in the nucleus, greatly affecting the differentiation pathway of a cell. It has been discovered as a co-suppressor of WT1 function (Wilms' Tumor suppressor protein 1) exerting its function by interacting with an N-terminal suppression domain of WT1 (Carpenter et al. 2004;Green et al. 2009). WT1 itself is a potent transcriptional regulator that activates or represses target genes including those for growth factors and regulators of cell division . Aberrant expression of WT1 is associated with several childhood and adult cancers (Rivera and Haber 2005;Yang et al. 2007). Additionally, a recent study discovered BASP1 to be downregulated in v-Myc-transformed chicken fibroblasts. Strikingly, ectopic expression of BASP1 renders fibroblasts resistant to subsequent cell transformation by v-Myc and it has been shown that the inhibition of v-Myc-induced cell transformation by BASP1 affects the transcriptional regulation of Myc target genes (Hartl et al. 2009). Other findings, reporting the frequent down-regulation of BASP1 expression in ALL (acute lymphocytic leukaemia) and CLL (chronic lymphocytic leukaemia) (Yeoh et al. 2002;Wang et al. 2004), as well as apoptosis-induced cleavage of BASP1 and its subsequent translocation to the cytoplasm (Ohsawa et al. 2008), again highlight the importance of BASP1 in transcription regulation.
To provide molecular information about this potential tumour suppressor protein we have started the NMR structure determination of recombinant human BASP1. The near complete chemical shift assignment reveals that BASP1 belongs to the class of intrinsically disordered proteins.

Protein expression and purification
The coding region for hBASP1 (human BASP1) was amplified by PCR from the mammalian expression vector Flag-hBASP1-pTKX3 (Ohsawa et al. 2008) introducing a 5 0 NcoI and 3 0 NotI site. Subsequently the fragment was inserted in-frame into the NcoI and NotI sites of the bacterial expression vector pET-M11 (Pinotsis et al. 2006), yielding pET-M11-hBASP1, encoding hBASP1 fused to an N-terminal His6-tag plus the TEV-cleavage site (H6-hBASP1). 15 N/ 13 C labeled H6-hBASP1 was expressed in the E. coli strain Rosetta(DE3)pLysS following a new expression protocol for efficient isotopic labeling of recombinant proteins using a fourfold cell concentration in isotopically labeled minimal medium (Marley et al. 2001). The cells were collected after 4 h of expression at 37°C by centrifugation at 5,000 rpm for 15 min and resuspended in 40 ml of ice-cold lysis buffer (20 mM Na x H (3-x) PO 4 , 50 mM NaCl, 10 mM imidazole, pH 7.2) per liter of the original bacterial culture. Bacteria were lysed by passing through a French press, and the cell lysate was cleared by centrifugation at 18,000 rpm for 20 min. The supernatant containing the soluble protein fraction was loaded onto a Ni 2? loaded HiTrap 5 ml affinity column (GE Healthcare), washed with 2 column volumes of high salt buffer (20 mM Na x H (3-x) PO 4 , 1.5 M NaCl, 10 mM imidazole, pH 7.2) and eluted with high imidazole buffer (20 mM Na x H (3-x) PO 4 , 50 mM NaCl, 0.5 M imidazole, pH 7.2) using a linear gradient of 15 column volumes. The H6-hBASP1 containing fractions were collected and the buffer was exchanged by 4 steps of concentration in an Amicon Ultra-15 centrifugal filter device 10 K NMWL (Amicon) and subsequent dilution in target buffer (20 mM Na x H (3-x) PO 4 , 50 mM NaCl, pH 6.0). NMR samples contain 1.5 mM uniformly 15 N/ 13 C labeled protein in 20 mM sodium phosphate (pH 6.0, in 90 % H 2 O and 10 % D 2 O), 50 mM NaCl and 0.2 % sodium azide.

NMR experiments
All spectra were acquired at 298 K on an Agilent Direct Drive 700 MHz spectrometer using the standard 5 mm 1 H-13 C-15 N triple-resonance probe head.

Extent of assignment and data deposition
The 1 H-15 N HSQC spectrum of H6-hBASP1 shows a very narrow peak dispersion in the 1 H dimension typical for intrinsically disordered proteins (Fig. 1). Extensive signal overlap in conventional 2D & 3D spectra could be overcome by using the aforementioned 5D experiments. 99 % of backbone 15 N, 99.5 % of 1 H N , 96.5 % of 13 C a , 74 % of 1 H a , 86.2 % of 13 C b , 81.4 % of 1 H b and 98.7 % of 13 C 0 resonances have been assigned. Additionally, H(CC-tocsy) CONH spectra allowed the assignment of several sidechain atoms. Figure 2 shows sample strips of sequential resonance assignment in a 5D (HACA)CON(CA)CONH and HN(CA)CONH experiment. Secondary chemical shifts for 13 C 0 , 13 C a , 1 H a (Fig. 3) show only minor deviations from random coil chemical shift values. Interestingly, the N-terminus appears to harbour stretches with slight a-helical structure propensities, whereas the rest of the a b c Fig. 3 Secondary chemical shifts for a 13 C 0 , b 13 C a , and c 1 H a using sequence-specific random coil chemical shifts of intrinsically disordered proteins (Tamiola et al. 2010) a b Fig. 2 2D spectral planes for consecutive amino acids in H6-hBASP1 obtained by SMFT processing of the 5D randomly sampled signal. 2D cross-sections of a 5D (HACA)CON(CA)CONH (N i -CO i-1 & N i-1 -CO i-2 ) and b 5D HN(CA)CONH (HN i -N i & HN i?1 -N i?1 ) protein seems to adopt a rather extended conformation indicated by positive 1 H a chemical shift differences.
The 1 H, 13 C and 15 N chemical shifts have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu/) under the BMRB accession number 18417.