Diagram illustrating predicted structure of the otoferlin protein (GenBank™ AAG12991, human). The SMART program (EMBL, Heidelberg, Germany) was used to predict the six C2 domains, A (residues 2–97), B (255–353), C (418–529), D (961–1068), E (1493–1592), F (1733–1863), and the carboxyl-terminal transmembrane region (residues 1964–1986). The large arrowheads indicate the positions of two mutations that were the focus of the present study, in domains C2D (1011 human; 1010 mouse) and C2F (1825). The four thick arrows relate to other single amino acid substitutions known to cause deafness (left to right): P490Q, I515T, R1939Q, and P1987R (12, 13). The thin arrows correspond to points of truncation: Arg²³⁷, Glu⁵⁵¹, Arg⁷⁰⁸, Gln⁸²⁹, Trp¹⁴²⁵, and Tyr¹⁴⁹⁷ (12). Mouse otoferlin sequence (NM_031875) is 98% identical to human sequence and exactly the same length (1997 amino acids).

Single amino acid substitution (Pro-Ala) in otoferlin C2F diminishes syntaxin 1A binding. A, SPR analysis was performed with purified native C2F (upper trace) and mutated C2Fm (lower trace) fusion peptides, injected as analytes versus immobilized syntaxin 1A or reference (surface blocked with ethanolamine, not shown), respectively. B, bars 1 and 2 illustrate average maximum response of previous upper and lower traces, respectively. C, GST pull-down assay showing interaction of GST-syntaxin 1A fusion peptide with otoferlin native C2F and mutated C2Fm fusion peptides carried out in the same experiment. Lane 1, molecular mass marker (same as for Fig. 2B); lane 2, GST-syntaxin 1A + C2F; lane 3, GST-syntaxin 1A + C2Fm; lane 4, GST + C2F control. D, secondary structural prediction for otoferlin C2F domain amino acid sequence (NM_031875), performed using the SSpro (21) program. Otoferlin C2F exhibits type II C2 topology (30), with seven strands of predicted β structure (strands 1–7, arrows and lines above sequence), one strand of predicted helical structure (strand 8), and intervening loops of extended (random coil) structure (21). Tryptophan residues that might increase intrinsic fluorescence of C2F when folded correctly are underlined. Letters at Pro-Ala mutation site (thought to reduce Ca²⁺ affinity of otoferlin C2F) are in bold. Four carboxylic amino acid residues, positioned to possibly coordinate Ca²⁺ binding (22), are shaded, and other nearby carboxylic residues are marked with dots. Predictions for turns in the secondary structure are marked by ∼ and bends by ^. The lowest line shows consensus amino acids with identity of 50% or more, as derived from sequence alignment of 65 different C2 domains (22), with hyphens indicating nonconsensus residues. E, normalized fluorescence spectra for affinity-purified C2Fm domain. The Pro-Ala mutated C2Fm domain protein (20 μm concentration) is insensitive to Ca²⁺, with no difference in the fluorescence spectrum between conditions of 61 μm Ca²⁺ (solidtrace) and EGTA(dashed trace). F, normalized fluorescence spectra for affinity-purified native C2F domain. When 61 μm Ca²⁺ is added to the native C2F protein solution (20 μm), the fluorescence is considerably elevated (solid trace), compared with lowered fluorescence for the same solution with added 2 mm EGTA (dashed trace). G, circular dichroism spectral analysis of the C2F domain fusion proteins purified with urea buffer followed by dialysis against PBST buffer (thick trace; see text) versus CD spectrum for C2F purified under nondenaturing conditions, in PBST (dashed trace).

Otoferlin C2D-calcium channel interaction. A, yeast two-hybrid assay indicates otoferlin C2D binding to the Ca_v1.3 II-III loop. Lane 1, C2D bait vector + II-III loop prey vector; lane 2, C2D bait vector + empty prey vector. B, pull-down assay showing interaction of GST-Ca_v1.3 II-III loop with otoferlin C2D (native) and C2Dm (mutated) fusion peptides. The lanes marked S show the molecular mass standards; lanes 1 and 2 are negative controls (glutathione beads alone + C2D, GST lysate + C2D, respectively). Lane 3 indicates no pull-down of C2Dm with the GST-Ca_v1.3 II-III loop, whereas lane 4 shows an expected band of ∼27 kDa representing C2D bound to GST-Ca_v1.3 II-III loop. C, SPR analysis of Ca_v1.3-C2D interaction. Using a sensor chip containing immobilized Ca_v1.3 II-III loop, we examined the interaction of the otoferlin C2D domain and its mutated form C2Dm with the Ca_v1.3 II-III loop. The SPR sensorgram shows maximum binding of ∼200 RU for 250 nm C2D (61 μm free Ca²⁺). There was a marked decrease in the binding when the mutated C2Dm domain (250 nm) was analyzed under the same conditions. D, C2D-Ca_v1.3 II-III loop interaction is Ca²⁺-sensitive, and mutation C2Dm significantly reduces the Ca²⁺ sensitivity. With Ca_v1.3 II-III loop as ligand, a series of free Ca²⁺ concentrations was tested, with C2D (dotted bars) and C2Dm (clear bars) as analytes (250 nm). Free Ca²⁺ concentration was adjusted to 0 (i.e. 1mm EGTA), 44, 61, 95, 199, and 371 μm (bars 1–6, respectively).

Interaction of otoferlin C2F and C2A domains with syntaxin 1A. A, yeast two-hybrid assay (replicate streakings) showing C2F binding to syntaxin 1A. Lane 1, syntaxin 1A prey vector + empty bait vector (negative control); lane 2, syntaxin 1A prey vector + C2F bait vector. The results for empty prey vector + C2F bait vector (not illustrated) were similar to those shown in lane 1. B, Western blot for GST pull-down assay, showing interaction of GST-syntaxin 1A fusion protein with otoferlin C2F fusion proteins. Lane 1, C2F + bacterial lysate without GST or GST-syntaxin 1A (negative control); lane 2, molecular mass standard (23, 34, 43, and 55 kDa, bottom to top); lane 3, C2F + GST-syntaxin 1A; lane 4, C2F + bacterial lysate with GST only (negative control). C, SPR analysis of otoferlin C2F binding to syntaxin 1A (upper trace). Association and dissociation phases can be discerned. Affinity-purified syntaxin 1A histidine-tagged fusion peptide, immobilized on the SPR sensor chip, served as the ligand, with C2F as the analyte. The response was recorded as the RU for the ligand minus the RU for the reference. Blank buffer with 10 μg/ml bovine serum albumin, run under the same conditions, served as a negative control (lower trace). D, Ca²⁺ dependence of otoferlin C2F interaction with syntaxin 1A. With syntaxin 1A as ligand, 200 nm of C2F domain fusion peptide, dissolved in a series of HBS-P buffers representing free Ca²⁺ concentrations of 0 (i.e. 3 mm EGTA), 30, 33, 37, 40, 44, 61, 95, 199, and 371 μm (bars 1–10, respectively) was analyzed for binding by SPR. Each bar shows maximum binding (RU). (All bar graphs in this figure and in subsequent figures indicate RU ± S.E., n = 3.) E, SPR time course showing calcium independence of otoferlin C2A-syntaxin 1A interaction. The upper trace represents the absence of Ca²⁺ (3 mm EDTA), and the lower trace the presence of Ca²⁺ (44 μm). Syntaxin 1A served as ligand and C2A (200 nm) as analyte. F, SPR response maxima illustrating calcium independence of otoferlin C2A-syntaxin 1A interaction. The analyte solution contained 1 mm EGTA (bar 1) or 61 μm free Ca²⁺ (bar 2). Injection of HBS-E buffer alone containing 1 mm EGTA plus 61 μm free Ca²⁺ served as a negative control (bar 3). Ten μg/ml bovine serum albumin was injected as another negative control (bar 4).

SNAP-25-C2F interaction. A, SPR analysis of C2F and C2Fm interaction with SNAP-25. Purified C2F and C2Fm fusion peptides (100 nm) were injected as analytes. B, Ca²⁺ sensitivity of C2F binding to SNAP-25. SNAP-25 was immobilized as ligand and C2F domain fusion peptide (100 nm) served as analyte in buffer representing a series of free Ca²⁺ concentrations. Bars 1–10 represent, respectively, 0 (i.e. 3 mm EGTA), 30, 33, 37, 40, 44, 61, 95, 199, and 371 μm Ca²⁺. Each bar shows the maximum binding (RU) obtained.

Quantitative otoferlin C2 domain SPR interaction kinetic curves (see summary of kinetic constants in Table 2). A, syntaxin 1A interaction with the otoferlin C2F domain. Purified syntaxin 1A was immobilized on a CM5 sensor chip, and C2F was diluted in a series of concentrations (0, 25, 50, 75, and 100 nm, bottom to top). The curves were selected and fit using a global fitting method and a 1:1 Langmuir binding model (not illustrated) with drifting base line (17). Deviation of fit from model, along the curves, is indicated by the residual (RES) plots for association (0–300 s) and dissociation (300–650 s). B, SNAP-25 interaction with the otoferlin C2F domain. The curves represent C2F concentrations of 0, 40, 80, 160, and 320 nm, evaluated as in A. C, Ca_v1.3 II-III loop interaction with the otoferlin C2D domain. The purified Ca_v1.3 II-III loop fusion protein was immobilized as before, and C2D analyte was tested for interaction at 0, 75, 150, 300, and 600 nm concentrations.