Structural organization of the nine spectrin repeats of Kalirin

Abstract

Sequence analysis suggests that KALRN, a Rho GDP/GTP exchange factor genetically linked to schizophrenia, could contain as many as nine tandem spectrin repeats (SRs). We expressed and purified fragments of Kalirin containing from one to five putative SRs to determine whether they formed nested structures that could endow Kalirin with the flexible rodlike properties characteristic of spectrin and dystrophin. Far-UV circular dichroism studies indicated that Kalirin contains nine SRs. On the basis of thermal denaturation, sensitivity to chemical denaturants, and the solubility of pairs of repeats, the nine SRs of Kalirin form nested structures. Modeling studies confirmed this conclusion and identified an exposed loop in SR5; consistent with the modeling, this loop was extremely labile to proteolytic cleavage. Analysis of a direpeat fragment (SR4:5) encompassing the region of Kalirin known to interact with NOS2, DISC-1, PAM, and Arf6 identified this as the least stable region. Analytical ultracentrifugation indicated that SR1:3, SR4:6, and SR7:9 were monomers and adopted an extended conformation. Gel filtration suggested that ΔKal7, a natural isoform that includes SR5:9, was monomeric and was not more extended than SR5:9. Similarly, the nine SRs of Kal7, which was also monomeric, were not more extended than SR5:9. The rigidity and flexibility of the nine SRs of Kal7, which separate its essential N-terminal Sec14p domain from its catalytic domain, play an essential role in its contribution to the formation and function of dendritic spines.

Fig. 1. Kalirin proteins examined

A. The domain structures predicted for Kal7 and ΔKal7, a naturally occurring splice variant, are shown; SR6 and SR8, which are not recognized as spectrin-like repeats by SMART, are stippled. The catalytic Dbl homology (DH) and adjacent pleckstrin homology (PH) domain form the GDP/GTP exchange factor domain. The C-terminus of Kal7 is a Type 1 PDZ binding motif, Ser-Thr-Tyr-Val. B. The SR proteins indicated were expressed as GST fusion proteins, cleaved and purified. C. The seven purified SR proteins studied (1 μg of each) were subjected to SDS-PAGE, transferred to a PVDF membrane and visualized using Coomassie Brilliant Blue 250. The mass of the molecular weight markers is indicated, as is the molecular weight (M_w) predicted for each SR protein.

Fig. 2. Circular dichroism – SR1:3

A. The far UV CD spectrum for SR1:3 is shown. B. The fraction of SR1:3 that was unfolded during a thermal denaturation experiment monitored at 222nm is shown. Data from multiple analyses of SR1:3 are summarized in Table 2.

Fig. 3. Circular dichroism – SR4:5, SR5:6 and SR4:6

The far UV CD spectra for SR4:5, SR5:6 and SR4:6 and the fraction of each protein that was unfolded during a thermal denaturation experiment monitored at 222 nm are shown. Data from multiple analyses of SR4:5, SR5:6 and SR4:6 are summarized in Table 2.

Fig. 4. Circular dichroism – SR4:7, SR7:9 and SR5:9

The far UV CD spectra for SR4:7, SR7:9 and SR5:9 and the fraction of each protein that was unfolded during a thermal denaturation experiment monitored at 222 nm are shown. Data from multiple analyses of SR4:7, SR7:9 and SR5:9 are summarized in Table 2.

Fig. 5. Sensitivity to chemical denaturants

SR4:5, SR5:6, SR4:6 and SR4:7 were incubated in guanidine HCl (0 to 4.0 M) or urea (0 to 6.0 M) for 12h at 27°C and then subjected to far UV CD analysis. The fraction of each protein unfolded was assessed by monitoring the signal at 222 nm. Data for the different SR proteins are summarized in Table 3.

Fig. 6. Results of molecular modeling

A. The sequences of the three tri-repeat SR proteins were submitted to the automated I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I TASSER/). Each SR is indicated by a different color; the linker regions connecting adjacent repeats are shown in red (SR1:3 and SR7:9) or blue (SR4:6). B. The backbone structures predicted for the di-repeat SR4:5 protein are shown in an orientation that highlights the helical region in the B/C linker. C. Purified GST-SR4:7 was incubated with immobilized TPCK trypsin (Pierce) for the times indicated, fractionated by SDS-PAGE and visualized using antiserum to SR4:7. Only the top and bottom bands were visualized using antibody to GST; the antiserum used was not affinity-purified and recognizes both SR4:7 and GST. A larger amount of GST-SR4:7 was treated with immobilized trypsin for 30 min and fractionated by SDS-PAGE. The Coomassie stained bands (#1, #2, #3) indicated by black arrows were excised from the PVDF membrane and subjected to Edman degradation using an Applied Biosystems Model 477A pulsed-liquid sequencer with on-line HPLC identification; the cleavage sites (blue arrows) and sequences obtained are shown.

Fig. 7. Revised identification of helix and loop regions in the spectrin repeat region of Kalirin

The A, B and C helices of the nine spectrin repeats of Kalirin identified by molecular modeling are indicated. Pro residues are depicted in green. Helical regions predicted within the A/B and B/C loops are shown in underlined Italics. The sequence of SR1 of human erythroid βI spectrin is shown for comparison.

Fig. 8. Analytical ultracentrifugation

SR1:3, SR4:5, SR5:6, SR4:6, SR4:7 and SR7:9 were subjected to analytical ultracentrifugation. Data for three concentrations of SR1:3, SR4:6 and SR7:9 are shown; there was no indication of reversible dimerization. The calculated mass for each SR protein analyzed indicates that it is monomeric. The frictional ratios determined (Table 4) indicate that each of these proteins is an extended rod.

Fig. 9. Gel filtration

SR proteins were analyzed on a Superose 6 column calibrated using the indicated globular protein standards. The elution volume for each globular protein is plotted against its published Stokes radius (red squares) . The elution volume for each SR protein subjected to analytical ultracentrifugation is plotted against its experimentally determined Stokes radius (green circles). Purified SR5:9 and lysates of non-neuronal cells transiently expressing Kal7 or ΔKal7 were analyzed on the same column; their elution volumes were determined by Western blot analysis of individual fractions. Based on their experimentally determined elution volumes, Stokes radii were estimated using the best fit line for the SR constructs.