Integrated protein array screening and high throughput validation of 70 novel neural calmodulin-binding proteins

Abstract

Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labeled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human neural proteins from a human brain cDNA library. We designed a screen to find high affinity (K(D) < or = 1 microm) binding partners of calmodulin and identified 76 human proteins from all intracellular compartments of which 72 are novel. We measured the binding kinetics of 74 targets with calmodulin using a high throughput surface plasmon resonance assay. Most of the novel calmodulin-target complexes identified have low dissociation rates (k(off) < or = 10(-3) s(-1)) and high affinity (K(D) </= 1 mum), consistent with the design of the screen. Many of the identified proteins are known to assemble in neural tissue, forming assemblies such as the spectrin scaffold and the postsynaptic density. We developed a microarray of the identified target proteins with which we can characterize the biochemistry of calmodulin for all targets in parallel. Four novel targets were verified in neural cells by co-immunoprecipitation, and four were selected for exploration of the calmodulin-binding regions. Using synthetic peptides and isothermal titration calorimetry, calmodulin binding motifs were identified in the potassium voltage-gated channel Kv6.1 (residues 474-493), calmodulin kinase-like vesicle-associated protein (residues 302-316), EF-hand domain family member A2 (residues 202-216), and phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (residues 400-415).

Fig. 1.

Scoring of human protein (hEx1) arrays with VisualGrid software. A, scoring pattern for each of 12 clones spotted in duplicate around a central guide dot. B, 1 μm CaM-Alexa488 binding to a field of the human protein array with positive clones highlighted in green squares on the blue grid that identifies each block of 24 spots.

Fig. 2.

Equivalent fields of high content protein array incubated overnight with 1 μm protein labeled with Alexa Fluor 488 in TBS buffer with 1 mm CaCl₂ followed by 6 × 10-min washes in TBST buffer for total of 60 min. A, calmodulin; B, secretagogin; C, calbindin D28k; D, calbindin D9k.

Fig. 3.

Surface plasmon resonance studies. A and B, schematics outlining the two SPR approaches with target proteins immobilized via His tag to Ni²⁺-NTA sensor chips (A) or calmodulin immobilized via a thiol linker to CM5 sensor chips (B). Shown are representative sensorgrams from SPR studies of calmodulin-target interactions in different kinetic ranges for calmodulin binding to His tag-immobilized ribosomal protein S2 (black), APLP1 (red), dynein (blue), and transcription factor IIIA (green) (C) and target protein binding to immobilized calmodulin for ZHX2 (black), elongation factor 2 (red), solute carrier family 16, member 8/MCT3 (blue), and semaphorin 4C (green) (D). All data were obtained in 10 mm Tris/HCl, 150 mm KCl, 1 mm CaCl₂, 0.005% (v/v) Tween 20, pH 7.5.

Fig. 4.

Western blots of immunoprecipitates from hippocampal cell lysates using either anti-calmodulin IgG (left lane in each panel) or anti-calbindin D9k IgG (right lane in each panel) in immunoprecipitation (IP) step and anti-glutamate (NMDA) receptor subunit ζ1 (A), anti-potassium voltage-gated channel Kv6.1 (B), anti-CaMKV (C), or anti-spectrin α chain IgG (D) in the immunoblotting (IB) detection step.

Fig. 5.

Ca²⁺ sensitivity of binding of calmodulin to calmodulin-interacting proteins on protein microarray. The microarray was incubated with anti-RGSHis₆ and Cy3-labeled anti-mouse IgG (A), 1 μm CaM-Alexa546 in 1 mm CaCl₂ (B), and 1 μm CaM-Alexa546 in 1 mm EDTA (C). Lane 1, diphosphomevalonate decarboxylase; lane 2, ribosomal protein S2; lane 3, dynein; lane 4, ZNF358; lane 5, CaM kinase II α; lane 6, buffer; lane 7, CaM-Alexa546.

Fig. 6.

Shown is isothermal titration calorimetry at 25 °C for peptides titrated from 200 or 400 μm solutions into 10 μm calmodulin in 10 mm Tris, 150 mm KCl, pH 7. 50 with either 1 mm CaCl₂ (a, d, e, and g) or 1 mm EDTA (b) or peptide titrated into buffer (c). An initial injection of 5 μl was followed by 29 injections of 10 μl of peptide solution with 5-min equilibration time between injections. The upper panels show the raw data. The lower panels show integrated heats versus molar ratio of peptide to protein, and the solid lines represent the best fit to the data using a 1:1 binding model. a and b, KVGCh(474–493) titrated into calmodulin in the presence of 1 mm CaCl₂ (a) or 1 mm EDTA (b). c, KVGCh(474–493) titrated into buffer with no calmodulin. d, EFHA2(202–216) titrated into calmodulin in the presence of 1 mm CaCl₂. e, CaMKV(302–316) titrated into calmodulin in the presence of 1 mm CaCl₂. d, PIP5K1C(400–415) titrated into calmodulin in the presence of 1 mm CaCl₂.