Ca2+-saturated calmodulin binds tightly to the N-terminal domain of A-type fibroblast growth factor homologous factors

Abstract

Edited by Karen FlemingVoltage-gated sodium channels (Na v s) are tightly regulated by multiple conserved auxiliary proteins, including the four fibroblast growth factor homologous factors (FGFs), which bind the Na v EF-hand like domain (EFL), and calmodulin (CaM), a multifunctional messenger protein that binds the Na V IQ motif. The EFL domain and IQ motif are contiguous regions of Na V cytosolic C-terminal domains (CTD), placing CaM and FGF in close proximity. However, whether the FGFs and CaM act independently, directly associate, or operate through allosteric interactions to regulate channel function is unknown. Titrations monitored by steady-state fluorescence spectroscopy, structural studies with solution NMR, and computational modeling demonstrated for the first time that both domains of (Ca 2+ ) 4 -CaM (but not apo CaM) directly bind two sites in the N-terminal domain (NTD) of A-type FGF splice variants (FGF11A, FGF12A, FGF13A, and FGF14A) with high affinity. The weaker of the (Ca 2+ ) 4 -CaM-binding sites was known via electrophysiology to have a role in long-term inactivation of the channel but not known to bind CaM. FGF12A binding to a complex of CaM associated with a fragment of the Na V 1.2 CTD increased the Ca 2+ -binding affinity of both CaM domains, consistent with (Ca 2+ ) 4 -CaM interacting preferentially with its higher-affinity site in the FGF12A NTD. Thus, A-type FGFs can compete with Na V IQ motifs for (Ca 2+ ) 4 -CaM. During spikes in the cytosolic Ca 2+ concentration that accompany an action potential, CaM may translocate from the Na V IQ motif to the FGF NTD, or the A-type FGF NTD may recruit a second molecule of CaM to the channel.

Assessing the Role of Calmodulin’s Linker Flexibility in Target Binding

“…Its primary sequence is identical across all vertebrates [ 2 ] and comprises two domains connected by a linker ( Figure 1 ). How CaM maintains binding selectivity towards its targets with affinities ( ) varying from nM [ 3 ] to μM [ 4 ] has been studied for decades [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. These studies have generated valuable insights into factors contributing to its binding selectivity, which includes hydrophobic residues in targets that interact with CaM, CaM’s conformational heterogeneity at the binding surface, and its Ca -binding sensitivity.…”

Navigating the intricacies of cellular machinery

“…Although an indirect interaction between Na V -associated FGF and CaM has been suggested for Na V 1.4, direct interaction and potential stoichiometry have not been explored until the Shea laboratory's extraordinary attention to detail brought the molecular mechanism into focus ( 6 , 7 , 8 ). The group has a strong history of bringing molecular studies to relevant physiological questions, including notably their description of the cooperativity of Ca 2+ binding by CaM, of import to CaM’s extensive substrates.…”

“…Mahling et al. have now demonstrated that the Na V complex has multiple CaM-binding sites with differing calcium-saturating preferences, accentuating the nuanced and extremely dynamic nature of the interactions ( 6 ).…”

“…They observed these interactions using fluorescence spectroscopy titrations and solution NMR and determined that both lobes of (Ca 2+ ) 4 –CaM directly bind both FGF sites, whereas apo-CaM does not. They found (Ca 2+ ) 4 –CaM binding to the aptly named CaMBD to range from about 5- to 44-fold higher than binding to the LTP, with variation among the FGF isoforms; the affinity of (Ca 2+ ) 4 –CaM for the FGF CaMBD ranged from FGF12A with a K d of 107 nM to FGF13A with a K d of 13 nM ( 6 ).

Figure 1 The Shea group’s investigations revealed two novel CaM ( blue ) interaction domains on FGFs ( purple ; domains labeled CaMBD and LTP) with the CaMBD showing high affinity for calcium-saturated CaM.

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“…Together, these data suggest that upon CaM binding of calcium, the change in conformation results in CaM’s dissociation from the Na V channel to reassociate with the proximal FGF. Alternatively, a second CaM molecule may be present in the complex ( 6 ). CaM, calmodulin; CaMBD, CaM-binding domain; FGFs, fibroblast growth factors; LTP, long-term inactivation particle; Na V s, voltage-gated sodium channels.…”