PYK2 senses calcium through a disordered dimerization and calmodulin-binding element

Momin, Afaque A.; Mendes, Tiago; Barthe, Philippe; Faure, Camille; Hong, SeungBeom; Yu, Piao; Kadaré, Gress; Jaremko, Mariusz; Girault, Jean-Antoine; Jaremko, Łukasz; Arold, Stefan T.

doi:10.1038/s42003-022-03760-8

PYK2 senses calcium through a disordered dimerization and calmodulin-binding element

Article
Open access
Published: 09 August 2022

Volume 5, article number 800, (2022)
Cite this article

Download PDF

You have full access to this open access article

Communications Biology

PYK2 senses calcium through a disordered dimerization and calmodulin-binding element

Download PDF

Afaque A. Momin ORCID: orcid.org/0000-0002-5058-9445^1,2,
Tiago Mendes³^na1,
Philippe Barthe⁴^na1,
Camille Faure³,
SeungBeom Hong^1,2,
Piao Yu^1,2,
Gress Kadaré³,
Mariusz Jaremko²,
Jean-Antoine Girault ORCID: orcid.org/0000-0002-7900-1705³,
Łukasz Jaremko² &
…
Stefan T. Arold ORCID: orcid.org/0000-0001-5278-0668^1,2,4

2253 Accesses
6 Citations
4 Altmetric
Explore all metrics

Abstract

Multidomain kinases use many ways to integrate and process diverse stimuli. Here, we investigated the mechanism by which the protein tyrosine kinase 2-beta (PYK2) functions as a sensor and effector of cellular calcium influx. We show that the linker between the PYK2 kinase and FAT domains (KFL) encompasses an unusual calmodulin (CaM) binding element. PYK2 KFL is disordered and engages CaM through an ensemble of transient binding events. Calcium increases the association by promoting structural changes in CaM that expose auxiliary interaction opportunities. KFL also forms fuzzy dimers, and dimerization is enhanced by CaM binding. As a monomer, however, KFL associates with the PYK2 FERM-kinase fragment. Thus, we identify a mechanism whereby calcium influx can promote PYK2 self-association, and hence kinase-activating trans-autophosphorylation. Collectively, our findings describe a flexible protein module that expands the paradigms for CaM binding and self-association, and their use for controlling kinase activity.

Calcineurin

Article Open access 28 August 2020

Regulation of Multifunctional Calcium/Calmodulin Stimulated Protein Kinases by Molecular Targeting

Activation of PKA via asymmetric allosteric coupling of structurally conserved cyclic nucleotide binding domains

Article Open access 04 September 2019

Introduction

Cellular signal transduction relies to a large extent on the capacity of multidomain protein kinases to sense, process, and transduce specific information provided by proteins, small molecules or ions. Focal adhesion kinase (FAK) and protein tyrosine kinase 2-beta (PYK2) are two closely related non-receptor protein tyrosine kinases that play vital roles in various cellular functions, including motility, adhesion, signalling, and gene expression^1,2,3,4,5,6. Further, they are important drivers of cancer cell invasiveness^7,8,9.

The PYK2-encoding PTK2B gene originated via the duplication and diversification of the FAK-encoding PTK2 gene in vertebrates¹⁰. Both protein paralogs have the same domain organisation: an N-terminal band 4.1, ezrin, radixin, moesin (FERM) domain, a tyrosine kinase domain, and a C-terminal focal adhesion targeting (FAT) domain (Fig. 1a). These domains are linked by flexible regions containing several short linear interaction motifs and sites for post-translational modifications, particularly phosphorylation^{2,5,11,12,13,14,15,16}. The three folded domains are markedly better conserved (47%–60% sequence identity) than the linker regions (20%–30% identity; Fig. 1a)¹⁶.

**Fig. 1: Identification of the CaM-binding site in PYK2.**

This shared structural composition results in a significant similarity in the activation mechanisms and biological functions of PYK2 and FAK. Indeed, both proteins have an autoinhibited conformation in which the FERM domain binds to the kinase domain, thereby inhibiting its catalytic function^17,18. The activation of kinase-dependent functions requires ligand-induced self-association (dimerisation or clustering) to trigger autophosphorylation in trans of a pivotal tyrosine (Y397 in FAK and Y402 in PYK2). This tyrosine is situated in the region linking the FERM and kinase domains^{16,19,20,21,22,23}. Once phosphorylated, this tyrosine and an adjacent proline-rich motif form a bidentate-binding site for the SH2–SH3 domain fragments of Src kinases (Src and Fyn)²⁴. This bidentate interaction activates Src kinases, which in turn contribute toward most of the catalytic activity associated with the catalytically active Src–FAK or Src–PYK2 complexes²⁰.

In line with their evolutionary history and preserved domain structure, FAK and PYK2 have overlap** cellular functions, for example in cellular adhesion^1,2,25, growth factor receptor signalling²⁶, kinase-independent nuclear effects on gene expression, and p53 degradation²⁷. However, PYK2 is also functionally distinct from FAK, particularly based on its unique role in Ca²⁺ sensing^11,28,29. This role allows PYK2 to partake in signalling pathways triggered by elevated Ca²⁺ levels in cells, such as cellular stress initiated by extracellular signals^16,30,31. PYK2 acts as a central transducer of Ca²⁺ signals at cell contacts²⁹ and in other loci³². In contrast to FAK, which is found in many cell types and is abundantly expressed during development, PYK2 is highly enriched in the adult forebrain³³. In neurons, PYK2 is activated by Ca²⁺ influx following the depolarisation or activation of glutamate receptors^34,35,36. In particular, PYK2 activation by Ca²⁺ promotes neurite outgrowth in neurons and is required for synaptic plasticity³⁰. Moreover, PYK2 accumulates in the nucleus following Ca²⁺ influx³⁷, a step regulated by its phosphorylation³⁸. These differences in gene expression patterns, protein sequences, and Ca²⁺ sensing limit the functional redundancy of FAK and PYK2, and give rise to distinct and even antagonistic functions^{9,Full size image}

Dilution of [¹³C,¹⁵N] PYK2 KFL_728–839 from 100 to 10 µM, monitored using the 2D [¹H-¹⁵N] HSQC spectra, revealed selective peak broadening and chemical shift perturbations (CSPs) as the percentage of PYK2 KFL_728-839 molecules in the dimer decreased from 94% to 82%, in accordance with the dimerisation K_d of ~0.8 µM (Fig. 4b, top. Supplementary Fig. 9). The regions of significant CSP changes broadly correlated with an analysis of the peak intensity change as a result of sample dilution (Fig. 4b, bottom). Both methods revealed that the dilution affected many regions relatively weakly, and that these regions were scattered across the length of the molecule. We concluded that PYK2 KFL_728-839 dimerisation is a fuzzy and rather unspecific process.

The 17 significantly altered backbone amide signals (>interquartile range of CSPs) corresponded to residues that were evenly scattered along PYK2 KFL_728–839 (Fig. 4b, c and Supplementary Table 3). These 17 residues did not display a clear physicochemical bias; seven were polar, six were hydrophobic, two were positively charged and one was negatively charged. Only residues K803, M804, I807, and L808 formed a connected, predominantly hydrophobic surface patch on the helical region, suggesting the particular importance of these residues for dimerisation (Fig. 4c).

To address the possibility that an intermediate exchange regime concealed the resonances of the bound state at the 700 MHz frequency used, we also recorded the 100 µM [¹⁵N]-PYK2 KFL_728–839 sample at 950 MHz. We neither observed new signals appearing, nor a significant narrowing of signals at 950 MHz, nor significant modulations in the peak intensity ratio between measurements at 700 and 950 MHz, supporting that our analysis was not affected by a significant contribution of intermediate exchange (Fig. 4d and Supplementary Fig. 10).

In further support of the dynamic and fuzzy dimerisation mode, ¹H/¹³C chemical shifts assigned for the residues of the same type, including side-chain methyl groups, were similar. In agreement with the modest signal dispersion on 2D [¹H-¹³C] and [¹H-¹⁵N] HSQC spectra, we detected only sequential i + 1,3,4 nuclear Overhauser effects (NOEs) within the helix and i ± 1 NOEs elsewhere from the 3D ¹³C- and ¹⁵N-edited NOE spectroscopy (NOESY)–HSQC spectra (Supplementary Fig. 8b). Finally, we did not observe any intermolecular NOEs in ¹³C/¹⁵N-filtered NOE experiments performed on a mixture of [¹³C-¹⁵N]-labelled and unlabelled PYK2 KFL_728–839 at two pH values (6.5 and 7.5). Taken together, these observations confirm the fuzzy dimerisation mode of PYK2 KFL_728-839, in which several structural conformations and inter-chain contacts are constantly in a dynamic exchange (Supplementary Fig. 8c).

PYK2-KFL binds to CaM in a process involving more than one linear peptide association

As a next step, we used NMR to map the interaction sites between PYK2 KFL_728–839 and CaM. We titrated [¹³C,¹⁵N]-PYK2 KFL_728–839 with unlabelled CaM and, in a second series, assigned [¹³C,¹⁵N]-CaM (Supplementary Fig. 11a, b) and titrated it with unlabelled PYK2 KFL_728–839. In agreement with the results of our other binding experiments, we observed an interaction between PYK2 KFL_728–839 and CaM in the absence and the presence of Ca²⁺ (Fig. 5a–d and Supplementary Fig. 11c–f). In the Ca²⁺-free association, twice as many resonances from the CaM C-terminal lobe (16 residues) than from the N-terminal lobe (7 residues) significantly broadened beyond the detection or shifted their ¹H-¹⁵N resonances (Supplementary Table 4). In PYK2 KFL_728–839, most of the affected residues clustered within the helical region (residues 791–812) and flanking residues (R790, K813, M815; Fig. 5a, b). For both CaM and PYK2 KFL_728–839, the Ca²⁺-free association prominently involved charged residues, with good overall charge complementarity between the nine negative charges from CaM (seven from its C-lobe) and seven positive charges from PYK2 KFL_728–839. Additional marked changes occurred in resonances from hydrophobic residues (CaM: 9; PYK2 KFL_728–839: 14) (Fig. 5a, b, e, f and Supplementary Table 4).

**Fig. 5: NMR map** of the residues contributing to the CaM–PYK2 KFL association.**

In the presence of Ca²⁺, CaM [¹H-¹⁵N] backbone amide resonances were lost due to line broadening starting from the Ca²⁺/CaM:PYK2 KFL_728–839 ratio of 1:0.5. All CaM resonances disappeared at the ratio of 1:4, as expected for the >70 kDa protein complex resulting from a 2:2 interaction between Ca²⁺/CaM and PYK2 KFL_728–839 (Fig. 5c, d and Supplementary Fig. 11f). We identified the most strongly interacting residues in CaM as those in which the resonances broadened out or decreased below a threshold at Ca²⁺/CaM:PYK2 KFL_728–839 ratios up to 1:1. At the 250 µM concentration used for each of the proteins, and given the association K_d of ~1 µM, ~96% of the labelled Ca²⁺/CaM was bound to PYK2 KFL_728–839. Although the map** cannot be done with high confidence based on these data, considerably more hydrophobic CaM residues appear to be involved compared with the findings for titrations without Ca²⁺. Those residues were mostly contributed by the CaM N-lobe (Fig. 5g, h).

With Ca²⁺, the imprint of unlabelled Ca²⁺/CaM was also spread more broadly across [¹³C,¹⁵N]-PYK2 KFL_728–839. However, even at the highest Ca²⁺/CaM concentration, line broadening was not as severe as that observed for [¹³C,¹⁵N]-CaM titrated with PYK2 KFL_728–839, supporting that PYK2 KFL_728–839 retains significant flexibility when bound to Ca²⁺/CaM (Supplementary Fig. 11d). The presence of Ca²⁺ markedly increased the involvement of hydrophobic, polar, and acidic residues in PYK2 KFL_728–839. These additional residues were predominantly located within the regions outside the KFL helix, while the contributions from the helix region remained comparable in titrations with or without Ca²⁺ (Fig. 5c, d and Supplementary Table 4).

We concluded that without Ca²⁺, CaM and PYK2 KFL_728–839 form a charge-dominated association, largely based on the CaM C-lobe and PYK2 KFL_728–839 helix region. The addition of Ca²⁺ promotes more hydrophobic and polar interactions between additional regions on both proteins while largely, but not entirely, preserving the interactions occurring without Ca²⁺. These additional interactions enhance the binding affinity and markedly constrain the dynamics of CaM, but not of PYK2 KFL_728–839, as judged from line broadening.

The effect of Ca²⁺ on the association is in agreement with the previously reported mechanism that Ca²⁺ leads to structural changes in CaM that expose more hydrophobic residues, enabling stronger ligand binding⁴⁵. Hence, the ligands that bind to CaM in the absence of Ca²⁺ tend to form a weaker charge-based association, particularly with the CaM C-lobe⁴⁵. We noted that the CaM regions involved in PYK2 KFL_728–839 binding in the absence of Ca²⁺ are reminiscent of those mediating Ca²⁺-independent associations with an IQ motif (named after the first two amino acids commonly found in this consensus)⁴⁶ (Supplementary Fig. 11g). However, as shown above, the IQ motif alone did not bind to CaM, demonstrating that the association between CaM and PYK2 KFL_728-839 was more complex, in line with our NMR-binding site map**. Finally, we observed a marked overlap between the residues identified as contributing to dimerisation and those contributing to CaM binding, suggesting that both events are functionally linked. Noteworthy exceptions were both tryptophans (W797 and W821) that were only involved in CaM binding (Fig. 5i). Among FAK and PYK2 sequences, the residues involved in PYK2 KFL dimerisation are more conserved than those implicated in CaM binding, in agreement with dimerisation being a conserved FAK/PYK2 function and CaM binding being unique to PYK2 (conservations scores are 0.65 and 0.53, respectively, while the average conservation score of the KFL region is 0.56, where 0 is not conserved and 1 is completely conserved).

PYK2 control through the fuzzy dimeric CaM-binding element

We designed a series of experiments to probe the functional implications of our results. Given that Ca²⁺ influx promotes PYK2 self-association and Y402 autophosphorylation in cells^9,20,21, we tested whether there was synergy between CaM binding, PYK2 KFL dimerisation, and PYK2 autophosphorylation. Previous studies have provided evidence that PYK2 autophosphorylation, in response to G protein-coupled receptor stimulation, is prevented by CaM antagonists^47,48. Accordingly, we found that the CaM antagonist calmidazolium blocked the membrane depolarisation-induced increase of PYK2 autophosphorylation in PC12 cells (Fig. 6a). However, in vitro, we observed only a mild increase (~2.5-fold) in the dimerisation strength of PYK2 KFL_728–839 in the presence of Ca²⁺/CaM compared with the dimerisation strength of PYK2 KFL_728–839 alone (Fig. 6b). The weak effect of Ca²⁺/CaM binding on PYK2 KFL_728–839 dimerisation suggests the involvement of other factors in cellular full-length PYK2.

**Fig. 6: Control of PYK2 function through fuzzy CaM-binding and dimerisation of KFL.**

FAK and PYK2 self-associate only under specific cell conditions and in particular subcellular locations^20,21,22,23. Therefore, we examined whether, in the PYK2 monomer, KFL_728–839 interacts with other regions of the same PYK2 polypeptide. Our ITC and MST experiments revealed that monomeric PYK2 KFL_728–839 failed to bind to the FERM or FAT domains (Supplementary Fig. 12 a). However, monomeric PYK2 KFL_728-839 bound to the PYK2 FERM–kinase fragment with micromolar affinity (Fig. 6c). These observations suggest that when PYK2 is monomeric, PYK2 KFL_728-839 associates with the FERM–kinase fragment. This association may conceal the dimerisation and CaM-binding surface of the KFL as well as possible flanking motifs that mediate ligand interaction (e.g., PR2 and PR3) or phosphorylation (Fig. 6d).

Discussion

The hallmark difference between PYK2 and FAK is the capability of the former to sense and respond to increases in cellular Ca²⁺. Although there is a general agreement that the major (but not the only) mechanism for Ca²⁺ sensing is based on the recognition of Ca²⁺/CaM, its molecular mechanism remains a matter of debate^{16,40,6d, right panel). In a subcellular location with high local PYK2 concentration, Ca²⁺/CaM-enforced KFL dimerisation will stabilise the weak FERM–FERM domain interaction, while releasing the FERM–kinase fragment, thereby priming the protein for trans-autophosphorylation. In this dimeric CaM-bound conformation, the functional motifs on the KFL would be accessible, thereby leading to a catalytically active and ligand-accessible Src–PYK2 scaffold.}

CaM has already been shown to promote dimerisation in other ligands. For example, one CaM molecule can tether two molecules of the oestrogen receptor α or of the Na⁺/H⁺ exchanger NHE1 by simultaneously binding one molecule with each, its N- and C-terminal lobe, forming a 2:1 complex^66,67. However, conversely to these examples, CaM reinforces the already strong PYK2 KFL_728-839 dimer in a fuzzy 2:2 association.

Collectively, our results describe a flexible protein module that expands the structural and functional range for CaM binding and self-association. The features identified for this element in PYK2 may allow the discovery of other fuzzy interaction modules in eukaryotic proteomes. Our analysis also shows how PYK2 has adapted the mechanistic framework inherited from FAK for Ca²⁺ sensing. The enhanced understanding resulting from our work might inspire new approaches to control the activation and localisation of FAK and PYK2 for therapeutic purposes.

Methods

Cloning

The FAK and PYK2 DNA fragments FAK_764–845, FAK_776–841, FAK_804–832, PYK2_728–839, PYK2_761–839, and PYK2_789–839 from Homo sapiens were amplified using the oligonucleotide primers (IDT) summarised in Table 1. Cloning was performed using restriction digest with BamHI and XhoI and the constructs were ligated into a pET-32a (+) modified expression vectors.

Table 1 List of the primers used for cloning.

Full size table

Expression and purification

The N-terminal 6×His fusion protein constructs FAK KFL_776–841, FAK KFL_804–832, PYK2 KFL_728–839, PYK2 KFL_761–839 FAK FERM_31–405 PYK2 FERM_35-367, cloned in the modified pET-32a (+) vector, were expressed in the Escherichia coli and purified as described in⁶⁸, but with the following differences: Cells were induced with 0.5 mM IPTG instead of 0.3 mM; pH 8.0 was used instead of pH 7.0 in all buffers; no imidazole was added in the wash buffer for the nickel column purification, proteins were eluted from the nickel column using 500 mM imidazole instead of 250 mM; and eluted proteins were applied to a Superdex 75 size-exclusion column (GE Healthcare) using the SEC buffer containing 20 mM HEPES pH 7.5, 200 mM NaCl, and 2 mM DTT. The constructs of FAK KFL_776–841, FAK KFL_804–832, PYK2 KFL_728–839, PYK2 KFL_761–839 with an N-terminal wild-type MBP fusion tag were cloned into pETM44 vectors and expressed and purified as described previously⁴³, with the difference that the MBP tag was cleaved using TEV protease at a concentration ratio of 1:100 overnight at 4 °C; that the cleaved MBP tag was removed from the protein solution by passing it through amylose beads; and that eluted protein was applied to a Superdex 75 SEC column (GE Healthcare) using the SEC buffer. The eGFP-MLCK, eGFP-PYK2 KFL_728–839 and mScarlet-CaM constructs were cloned in a SUMOStar^TM vector with an N-terminal 6×His tag followed by a SUMO tag, and were purified using the protocol used for His-tagged proteins. The His and SUMO tags were cleaved by the ULP1 protease and were subjected to a Superdex 75 SEC (GE Healthcare) using SEC buffer. PYK2 FERM–kinase and human Src constructs were cloned into modified pFastBac vector with a N-terminal 6xHis–eGFP tag followed by a HRV-3C protease cleavage site⁶⁹. They were expressed in HighFive cells using the amplified virus from SF9 cells, which included the target genes generated by the Bac-to-Bac system. Cell lysate was incubated with 10 mL CNBr-activated Sepharose beads (GE Healthcare) coupled with anti-GFP nanobodies. The 6xHis–eGFP tag was cleaved by the HRV-3C protease at 4 °C overnight and the untagged protein was eluted by gravity from the GFP–nanobody column. Eluted proteins were further purified using Superose 6 increase 10/300 GL column (GE Healthcare) in SEC buffer. All proteins used in this study were stored at −80 °C in 20 mM HEPES pH 7.5, 150 mM NaCl, and 2 mM DTT buffer. Protein purity and molecular weights (Mw) were evaluated using SDS–PAGE gels, and protein identity was confirmed using mass spectroscopy. All the purification and solubility tags were proteolytically cleaved, unless mentioned otherwise. Synthetic peptides were purchased from GenScript.

Circular dichroism (CD)

CD spectroscopic experiments were conducted in biological triplicates at 25 °C using a spectropolarimeter (JASCO) with a 0.1-mm path-length cell. FAK KFL_776–841, PYK2 KFL_728–839, and CaM constructs were dialysed into PBS buffer (10 mM Na₂HPO₄, 2 mM KH₂PO₄, 30 mM NaCl, 0.5 mM TCEP, pH 6.5 and pH 7.5, respectively). PYK2 KFL_728–839, CaM, and PYK2 KFL_728–839: CaM complex samples were measured in the presence and absence of Ca²⁺. FAK KFL_776–841 was measured at 27 µM, PYK2 KFL_728–839 and CaM were measured at a concentration of 20 µM, whereas the PYK2 KFL_728–839: CaM complex was measured at 40 µM. FAK FERM_31–405 and PYK2 FERM_35-367 constructs were dialysed in 20 mM HEPES, 200 mM NaCl, 0.5 mM TCEP, pH 7.5. FAK FERM_31–405 was measured at 10 µM, and PYK2 FERM_35-367 was measured at 12 µM at 25 °C and 60 °C. CD spectra were recorded from 240 to 190 nm at an interval of 1 nm. The results were analysed using CAPITO⁷⁰ and given as CD in delta ellipticity (/M/cm).

Analytical size-exclusion chromatography (SEC)

SEC experiments were performed using the Superdex 200 30/300 column (GE Healthcare) on an AKTA pure system using a buffer containing 20 mM HEPES, 200 mM NaCl, and 2 mM DTT at pH’s 6.5 and 7.5. For CaM-binding experiments, the buffer was supplemented with 5 mM Ca²⁺ or 1 mM EGTA. The protein concentration for all samples was adjusted to 5 mg/mL and samples were pre-equilibrated for 1 h in the running buffer at 25 °C. Bio-Rad Mw standards were used. The Mw of each protein of interest was determined by dividing the elution volume of the standards and unknown by the void volume, which was 7.2 mL. The outputs were plotted against the log of the Mw of the standards. The SEC-MALS data was obtained with the protein passed through Superdex 200 30/300 column (GE Healthcare) in a buffer containing 20 mM HEPES, 200 mM NaCl, and 2 mM DTT at pH’s 6.5 and 7.5 through Agilent HPLC following DAWN MALS detector. Data was analysed using ASTRA software provided by the company.

Analytical ultracentrifugation (AUC)

AUC experiments were conducted at 25 °C using an ultracentrifuge (XL-I; Beckman) equipped with a 60 Ti rotor. 200 µL of 100 µM protein sample (MBP-fusion proteins and MBP alone at 1 mg/mL) in 20 mM HEPES, 150 mM NaCl, 2 mM DTT at pH 7.5 were centrifuged for 48 h. The rotor speeds were set at 6520, 15,777, and 23,263 × g. The partial specific volume of the protein, solvent viscosity, and solvent density were calculated using the company-provided software “Sednterp 1.09”⁷¹. Data were analysed using Sedphat 10.40⁷². The monomer–dimer equilibrium model was used to fit the sedimentation profiles using rotor stretch restraints and mass conservation.

Microscale thermophoresis (MST)

Recombinant purified 6×His–CaM, 6×His–PYK2 KFL_728–839, 6×His–PYK2 KFL_761–839, 6×His-FAK KFL_776–841, and 6×His–FAK KFL_804–832 were labelled using the NanoTemper His-tag labelling kit. Labelled proteins were kept at a concentration of 50 nM in 20 mM HEPES, 200 mM NaCl, 1 mM DTT, and 5 mM Ca²⁺ or 1 mM EGTA at pH 6.5. The unlabelled protein was serially diluted in the same buffer. For dimerisation experiments in the presence of CaM, CaM was added to each sample of the dilution series to reach a final CaM concentration of 10 μM. The measurements were performed in MST premium capillaries (NanoTemper Technologies, Germany) in 50% LED power and medium MST power. Measurements were performed in biological triplicates, except for the data shown in Fig. 6b, c, where technical triplicates were used. Data were analysed using the NT Analysis software.

Fluorescence anisotropy

Recombinant purified 6×His–CaM, 6×His–PYK2 KFL_728–839, 6×His–PYK2 KFL_761–839, 6×His-FAK KFL_776–841, and 6×His–FAK KFL_804–832 were labelled using the NanoTemper His-tag labelling kit. Labelled proteins were kept at a concentration of 50 nM in 20 mM HEPES, 200 mM NaCl, 1 mM DTT, and 5 mM Ca²⁺ or 1 mM EGTA at pH 6.5. The unlabelled protein was serially diluted in the same buffer. For dimerisation experiments in the presence of CaM, CaM was added to each sample of the dilution series to reach a final CaM concentration of 10 μM. Data were collected using spectraMax i3 (Molecular Devices) plate reader. The K_d value was fitted using single binding site mode and plotted using Prism 9.0 (www.graphpad.com).

Differential-scanning fluorimetry

The experiment was performed as previously described⁴³. Briefly, thermal stability for PYK2 KFL_728–839 and CaM was assessed in 20 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM EGTA and 1 mM DTT. FAK FERM_31–405 PYK2 FERM_35-367 was measured in 20 mM HEPES, pH 7.5, 200 mM NaCl, and 1 mM DTT with temperature ranging from 25 °C to 95 °C. All proteins were used at a concentration of 10 μM, and fluorescent dye SYPRO Orange was used at a final concentration of 5X.

Isothermal titration calorimetry (ITC)

The recombinant proteins were dialysed in 20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM DTT, and 5 mM Ca²⁺ or in 20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM DTT, and 1 mM EGTA and further degassed before using in ITC. Titrations were performed in biological triplicates on the MicroCal ITC200 instrument at 25 °C. Synthetic peptides and proteins in the 75 µL syringe were kept at a concentration of 500 µM (PYK2 FERM, FAK FERM, PYK2 FAT and FAK FAT) or 300 µM (CaM). These molecules were injected at a rate of 0.4 µL/injection into the 0.35 mL measurement cell Proteins in the measurement cell were kept at either 50 µM (CaM, PYK2 KFL_761–839, and FAK KFL_776–841) or 30 µM (PYK2 KFL_728–839). Control experiments were performed by injecting the same ligands into buffer. The Origin 7.0 software was used for data analysis.

Nuclear magnetic resonance (NMR)

Cells were grown with ¹⁵N-NH₄Cl and ¹³C₆H₁₂O₆ dissolved in M9 minimal medium solution, induced at OD_600nm = 0.7 with 500 µM IPTG and harvested after overnight incubation at 18 °C. Protein samples were purified, and NMR samples were prepared by dissolving the ¹⁵N-labelled protein in a 5% D₂O/95% H₂O solution in the conditions and concentrations described in Supplementary Table 5. NMR data were collected at 25 °C on the Bruker Avance III 950 MHz spectrometer equipped with a 5-mm z-gradient TCI CryoProbe. NMR samples contained ~0.25 mM ¹⁵N-labelled or ¹³C-¹⁵N-labelled protein dissolved in 20 mM HEPES buffer (pH 6.5), 150 mM NaCl, and 1 mM TCEP with 5% D₂O for the lock. ¹H chemical shifts were directly referenced to the methyl resonance of DSS, whereas ¹⁵N and ¹³C chemical shifts were referenced indirectly to the absolute ¹⁵N/¹H and ¹³C/¹H frequency ratios, respectively. All NMR spectra were processed using TopSpin (Bruker) and analysed using CINDY (http://abcis.cbs.cnrs.fr/CINDY/; Padilla, CBS). Backbone and side-chain resonance assignments were performed using standard 3D NMR experiments (HNCA, HNCACB, HN(CO)CA, CBCA(CO)NH, HNCO, HN(CA)CO, and [¹H¹⁵N] NOESY–HSQC). Changes in chemical shifts for analysing CSP data for ¹H and ¹⁵N were measured in ppm (ΔH and ΔN). The ¹⁵N shift changes were multiplied by a scaling factor of 0.2 and then the total change in CSP was calculated as follows: \(\left({{{{{{\mathrm{CSP}}}}}}i}\right)=\triangle {HN}=\sqrt{\frac{1}{2}\left[{\delta }_{H}^{2}+\left(\alpha .{\delta }_{N}^{2}\right)\right]}\) (1)⁷³. IQR values were calculated by the equation IQR = Q₁– Q₃ (2), where Q₁ is the first quartile, and Q₃ is the third quartile. The median values were calculated and data was plotted using Prism 9.0 (www.graphpad.com). To plot the 2D [¹H-¹⁵N] HSQC ratios of PYK2 KFL_728–839 peak intensities at 10 µM divided by those at 100 µM, the intensity values were retrieved by exporting data height with signal-to-noise values using NMRFAM-SPARKY⁷⁴. The intensity and signal-to-noise values for both concentrations of PYK2 KFL_728–839 were normalised according to the number of scans^75,76.

NOE cross-peaks identified on the NOESY spectra (mixing time 150 ms) were assigned via automated NMR structure calculations with CYANA 3⁷⁷. Backbone φ/ψ and side-chain χ1 torsion angles constraints were obtained from a database search procedure on the basis of backbone (¹⁵N, H_N, ¹³C′, ¹³Cα, Hα) and ¹³Cβ chemical shifts using TALOS-N⁷⁸. A total of 100 3D structures were generated using the torsion angle dynamics protocol of CYANA 3 from 374 NOEs, 18 hydrogen bonds, and 81 angular restraints (26 φ, 26 ψ, and 29 χ1) for PYK2 KFL_728–839. The 20 best structures (based on the final target penalty function values) were minimised using CNS 1.2 according to the RECOORD procedure⁷⁹.

Visible immunoprecipitation (VIP)

Purified recombinant eGFP-tagged PYK2 KFL_728–839 or the eGFP-tagged human myosin light chain kinase (MLCK) fragment RRKWQKTGHAVRAIGRLSS (taken from PDB 2K0F) were mixed with anti-GFP nanobody beads. For binding assays, mScarlet-CaM was mixed with the beads and incubated at 4 °C for 2 h in 20 mM HEPES, 150 mM NaCl, 1 mM DTT, 10 mM Ca²⁺ or 10 mM EGTA. The beads were thoroughly washed with wash buffer (20 mM HEPES, 150 mM NaCl, 1 mM DTT, 10 mM Ca²⁺ or 20 mM HEPES, 150 mM NaCl, 1 mM DTT, 10 mM EGTA) and then visualised under the red and green filters adapted to the excitation wavelengths of eGFP at 488 nm and mScarlet at 569 nm. The experiments were performed in the presence of 10 mM Ca²⁺ or of 10 mM EGTA.

Cell culture

For pulldown assay, HEK293T cells were seeded in 6-cm dishes, at a density of 400,000 cells per dish, and maintained in DMEM cell culture medium (Gibco), supplemented with 10% fetal bovine serum (Gibco) and 0.2% penicillin/streptomycin (Gibco), in a humidified atmosphere at 5% CO₂ and 37 °C. Cells were transiently transfected 24 h after seeding with 8 μL Lipofectamine 2000 (Invitrogen) per 4 µg plasmid DNA as previously described^37,38. Forty-eight h after transfection, cells were harvested with 300 μL ice-cold lysis buffer containing 50 mM Tris-base pH 8.0, 100 mM NaCl, 1 mM EGTA, 0.1%, v/v, Triton X-100, Complete Mini EDTA-free (Sigma) and PhosSTOP (Sigma). Cell membranes were disrupted by freeze-thaw, followed by pipetting, and protein extracts were cleared by centrifugation at 11,000 x g at 4 °C for 15 min. Protein concentration was quantified using the Micro BCA Protein Assay Kit (Thermo Scientific). For treatment with CaM antagonist, PC12 cells were grown on type I collagen-coated dishes (BD Biosciences) in RPMI medium (Gibco) containing 10% horse serum, 5% fetal calf serum. Transfection was performed as described above with GFP-PYK2 WT when cells were at about 70% confluency. Forty-eight hours after transfection, cells were treated with 50 µM calmidazolium (Cz) for 30 min, followed by depolarisation by isosmotic replacement of 40 mM NaCl by 40 mM KCl in the extracellular medium for 3 min, as previously described³⁷. For this procedure, half the medium was replaced by a solution containing 1 mM MgCl₂, 2 mM CaCl₂, 25 mM HEPES, and either 135 mM NaCl, (control solution), or 55 mM NaCl and 80 mM KCl (high [K⁺]). PC12 cells were lysed on ice, in RIPA buffer, followed by quantification, electrophoresis and immunoblot.

Pulldown assay and immunoblot

CaM-Sepharose 4B beads (Sigma) were left to equilibrate at room temperature (RT) for 1 h, mixed thoroughly and 50 μL beads slurry was dispensed in 2-mL centrifuge tubes. Beads were washed with pulldown buffer (50 mM Tris-base pH 8, 100 mM NaCl, Complete Mini EDTA-free, PhosSTOP) supplemented with either 2 mM CaCl₂ or 10 mM EGTA. Equal amounts of protein were added to the tubes containing the CaM beads (200 µg from each cell transfection; 100 μg from each recombinant purified protein), volume was adjusted to 1 mL with respective pulldown buffers, supplemented with CaCl₂ or EGTA, and the suspensions were incubated for 2.5 h at 4 °C with light agitation. The beads with bound proteins were collected by centrifugation at 18,000 x g for 10 min, followed by three washes with a buffer of the same composition as used in each incubation, and followed by incubation with 50 µL of opposite buffer and 10 µL 100 mg/mL SDS (Sigma) for 30 min at RT with light agitation. In parallel, 1% of protein from cell transfection or of recombinant purified protein were collected into clean centrifuge tubes. After the last incubation, 4x Laemmli buffer was added to input and beads fractions and samples were heated at 95 °C for 10 min. Input and bead (Ca²⁺ or EGTA) fractions from cell transfections were subjected to electrophoresis in denaturing conditions (SDS–PAGE) and transferred to nitrocellulose membranes for immunoblotting. Membranes were saturated in 25 g/L BSA in TBS with 0.1% Tween-20 (TBS-T), followed by washes with TBS-T, overnight incubation at 4 °C with primary antibody chicken anti-GFP (#A10262, Invitrogen) at 1:1000, washes with TBS-T, 1 h incubation at RT with primary antibody mouse anti-tubulin (#T9026, Sigma) at 1:10000, washes with TBS-T, 2 h incubation at RT with secondary antibodies anti-chicken 800 nm (#926-32218, LI-COR) and anti-mouse 680 nm (#926-68072, LI-COR), both at 1:5000, washes with TBS-T, and fluorescence acquisition with Odyssey CLx (LI-COR). Protein extracts from Cz treatment and depolarisation experiments were submitted to electrophoresis, but were immunoblotted with primary antibody rabbit anti-PYK2 (#P3902, Sigma) or primary antibody Rabbit anti-pY402 PYK2 (#44-618G, Invitrogen) at 1:1000, followed by secondary antibodies anti-rabbit 800 nm (#926-32213, LI-COR) at 1:5000. Protein band quantification was performed using Image Studio Lite 5.2 (LI-COR). For quantification of experiments of binding of GFP-PYK2 (WT or deleted forms) to CaM beads in the presence of Ca²⁺or EGTA, the amount of GFP-immunoreactivity associated with the beads was divided by the amount of GFP-immunoreactivity in the input for correction of the variable amounts of fusion protein depending on the constructs. For the experiments in which phosphorylation of PYK2 in response to depolarisation was studied in the absence or presence of Cz, pY402 immunoreactivity was divided by total PYK2 immunoreactivity. Input and bead (Ca²⁺ and EGTA) fractions from recombinant purified protein were submitted to electrophoresis, stained using InVision His-Tag In-Gel stain and observed using BioRad Gel dock imager at 260 nm.

Bioinformatics

The sequence alignment of the 78 sequences between FAK and PYK2 was performed using the online server MAFFT https://mafft.cbrc.jp/alignment/software/. The alignment result was visualised using Guidance 2.0 http://guidance.tau.ac.il/ver2/. Secondary structure predictions were made with PHD⁸⁰, PSIPRED⁸¹, JPred4⁸², and AlphaFold2⁸³, while coiled-coil prediction was made using COILS⁸⁴ and Waggawaga webserver for NCOILS⁸⁵. CaM-binding motifs were taken from cam.umassmed.edu⁸⁶.

Statistics and reproducibility

Statistical data analysis was performed using GraphPad Prism 9.0 and Microsoft Excel 16.62. The statistical data values are given as mean ± standard error (SD) of the mean, where ‘n’ is the number of replicates indicated in applicable figure legends. Curve fitting for binding experiments was performed using a one-site total non-linear fit.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

NMR assignments of PYK2 KFL_728–839 are deposited in the Biological Magnetic Resonance Data Bank (BMRB accession code: 50961). The source data underlying graphs, plots, and charts in the manuscript are presented in Supplementary Data.

References

Hanks, S. K., Calalb, M. B., Harper, M. C. & Patel, S. K. Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin. Proc Natl Acad Sci USA 89, 8487–8491 (1992).
Article CAS PubMed PubMed Central Google Scholar
Schaller, M. D. et al. pp125^FAK, A structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci USA 89, 5192–5196 (1992).
Article CAS PubMed PubMed Central Google Scholar
Schaller, M. D. Cellular functions of FAK kinases: insight into molecular mechanisms and novel functions. J. Cell Sci. 123, 1007–1013 (2010).
Article CAS PubMed Google Scholar
Arold, S. T. How focal adhesion kinase achieves regulation by linking ligand binding, localization and action. Curr. Opin. Struct. Biol. 21, 808–813 (2011).
Article CAS PubMed PubMed Central Google Scholar
Hall, J. E., Fu, W. & Schaller, M. D. Focal adhesion kinase: exploring Fak structure to gain insight into function. Int. Rev. Cell Mol. Biol. 288, 185–225 (2011).
Article CAS PubMed Google Scholar
de Pins, B., Mendes, T., Giralt, A. & Girault, J. A. The Non-receptor Tyrosine Kinase Pyk2 in brain function and Neurological and Psychiatric diseases. Front Synaptic Neurosci. 13, 749001 (2021).
Sulzmaier, F. J., Jean, C. & Schlaepfer, D. D. FAK in cancer: mechanistic findings and clinical applications. Nat. Rev. Cancer 14, 598–610 (2014).
Article CAS PubMed PubMed Central Google Scholar
Lipinski, C. A. et al. The tyrosine kinase pyk2 promotes migration and invasion of glioma cells. Neoplasia 7, 435–445 (2005).
Article CAS PubMed PubMed Central Google Scholar
Naser R, Aldehaiman A, Diaz-Galicia E, Arold ST. Endogenous control mechanisms of FAK and PYK2 and their relevance to cancer development. Cancers (Basel) 10, 196 (2018).
Corsi, J. M., Rouer, E., Girault, J. A. & Enslen, H. Organization and post-transcriptional processing of focal adhesion kinase gene. BMC Genomics 7, 198 (2006).
Article PubMed PubMed Central CAS Google Scholar
Lev, S. et al. Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions. Nature 376, 737–745 (1995).
Article CAS PubMed Google Scholar
Girault, J. A., Labesse, G., Mornon, J. P. & Callebaut, I. Janus kinases and focal adhesion kinases play in the 4.1 band: a superfamily of band 4.1 domains important for cell structure and signal transduction. Mol. Med. 4, 751–769 (1998).
Ceccarelli, D. F., Song, H. K., Poy, F., Schaller, M. D. & Eck, M. J. Crystal structure of the FERM domain of focal adhesion kinase. J. Biol. Chem. 281, 252–259 (2006).
Article CAS PubMed Google Scholar
Arold, S. T., Hoellerer, M. K. & Noble, M. E. The structural basis of localization and signaling by the focal adhesion targeting domain. Structure 10, 319–327 (2002).
Article CAS PubMed Google Scholar
Hayashi, I., Vuori, K. & Liddington, R. C. The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin. Nat. Struct. Biol. 9, 101–106 (2002).
Article CAS PubMed Google Scholar
Walkiewicz, K. W., Girault, J. A. & Arold, S. T. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions. Prog. Biophys. Mol. Biol. 119, 60–71 (2015).
Article CAS PubMed Google Scholar
Lietha, D. et al. Structural basis for the autoinhibition of focal adhesion kinase. Cell 129, 1177–1187 (2007).
Article CAS PubMed PubMed Central Google Scholar
Loving, H. S. & Underbakke, E. S. Conformational dynamics of FERM-mediated autoinhibition in Pyk2 tyrosine kinase. Biochemistry 58, 3767–3776 (2019).
Article CAS PubMed Google Scholar
Toutant, M. et al. Alternative splicing controls the mechanisms of FAK autophosphorylation. Mol. Cell Biol. 22, 7731–7743 (2002).
Article CAS PubMed PubMed Central Google Scholar
Park, S. Y., Avraham, H. K. & Avraham, S. RAFTK/Pyk2 activation is mediated by trans-acting autophosphorylation in a Src-independent manner. J. Biol. Chem. 279, 33315–33322 (2004).
Article CAS PubMed Google Scholar
Bartos, J. A. et al. Postsynaptic clustering and activation of Pyk2 by PSD-95. J. Neurosci. 30, 449–463 (2010).
Article CAS PubMed PubMed Central Google Scholar
Brami-Cherrier, K. et al. FAK dimerization controls its kinase-dependent functions at focal adhesions. EMBO J. 33, 356–370 (2014).
Article CAS PubMed PubMed Central Google Scholar
Goni, G. M. et al. Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes. Proc. Natl Acad. Sci. USA 111, E3177–E3186 (2014).
Article CAS PubMed PubMed Central Google Scholar
Arold, S. T. et al. The role of the Src homology 3-Src homology 2 interface in the regulation of Src kinases. J. Biol. Chem. 276, 17199–17205 (2001).
Article CAS PubMed Google Scholar
Sasaki, H. et al. Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily. J. Biol. Chem. 270, 21206–21219 (1995).
Article CAS PubMed Google Scholar
McLean, G. W. et al. The role of focal-adhesion kinase in cancer - a new therapeutic opportunity. Nat. Rev. Cancer 5, 505–515 (2005).
Article CAS PubMed Google Scholar
Lim, S. T. et al. Nuclear FAK promotes cell proliferation and survival through FERM-enhanced p53 degradation. Mol. Cell 29, 9–22 (2008).
Article CAS PubMed PubMed Central Google Scholar
Yu, H. et al. Activation of a novel calcium-dependent protein-tyrosine kinase. Correlation with c-Jun N-terminal kinase but not mitogen-activated protein kinase activation. J. Biol. Chem. 271, 29993–29998 (1996).
Article CAS PubMed Google Scholar
Hashido, M., Hayashi, K., Hirose, K. & Iino, M. Ca2+ lightning conveys cell-cell contact information inside the cells. EMBO Rep. 7, 1117–1123 (2006).
Article CAS PubMed PubMed Central Google Scholar
Girault, J. A., Costa, A., Derkinderen, P., Studler, J. M. & Toutant, M. F. A. K. and PYK2/CAKbeta in the nervous system: a link between neuronal activity, plasticity and survival? Trends Neurosci. 22, 257–263 (1999).
Article CAS PubMed Google Scholar
Avraham, H., Park, S. Y., Schinkmann, K. & Avraham, S. RAFTK/Pyk2-mediated cellular signalling. Cell Signal 12, 123–133 (2000).
Article CAS PubMed Google Scholar
Andreev, J. et al. Identification of a new Pyk2 target protein with Arf-GAP activity. Mol. Cell Biol. 19, 2338–2350 (1999).
Article CAS PubMed PubMed Central Google Scholar
Menegon, A. et al. FAK+ and PYK2/CAKbeta, two related tyrosine kinases highly expressed in the central nervous system: similarities and differences in the expression pattern. Eur. J. Neurosci. 11, 3777–3788 (1999).
Article CAS PubMed Google Scholar
Siciliano, J. C., Gelman, M. & Girault, J. A. Depolarization and neurotransmitters increase neuronal protein tyrosine phosphorylation. J. Neurochem. 62, 950–959 (1994).
Article CAS PubMed Google Scholar
Siciliano, J. C., Toutant, M., Derkinderen, P., Sasaki, T. & Girault, J. A. Differential regulation of proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) and pp125(FAK) by glutamate and depolarization in rat hippocampus. J. Biol. Chem. 271, 28942–28946 (1996).
Article CAS PubMed Google Scholar
Huang, Y. et al. CAKbeta/Pyk2 kinase is a signaling link for induction of long-term potentiation in CA1 hippocampus. Neuron 29, 485–496 (2001).
Article CAS PubMed Google Scholar
Faure, C. et al. Calcineurin is essential for depolarization-induced nuclear translocation and tyrosine phosphorylation of PYK2 in neurons. J. Cell Sci. 120, 3034–3044 (2007).
Article CAS PubMed Google Scholar
Faure, C., Ramos, M. & Girault, J. A. Pyk2 cytonuclear localization: mechanisms and regulation by serine dephosphorylation. Cell. Mol. life Sci. 70, 137–152 (2013).
Article CAS PubMed Google Scholar
**ong, W. & Parsons, J. T. Induction of apoptosis after expression of PYK2, a tyrosine kinase structurally related to focal adhesion kinase. J. Cell Biol. 139, 529–539 (1997).
Article CAS PubMed PubMed Central Google Scholar
Kohno, T., Matsuda, E., Sasaki, H. & Sasaki, T. Protein-tyrosine kinase CAKbeta/PYK2 is activated by binding Ca2+/calmodulin to FERM F2 alpha2 helix and thus forming its dimer. Biochem. J. 410, 513–523 (2008).
Article CAS PubMed Google Scholar
**e, J. et al. Analysis of the calcium-dependent regulation of proline-rich tyrosine kinase 2 by gonadotropin-releasing hormone. Mol. Endocrinol. 22, 2322–2335 (2008).
Article CAS PubMed PubMed Central Google Scholar
Schaller, M. D. Calcium-dependent Pyk2 activation: a role for calmodulin? Biochem. J. 410, e3–e4 (2008).
Article CAS PubMed Google Scholar
Momin, A. A., Hameed, U. F. S. & Arold, S. T. Passenger sequences can promote interlaced dimers in a common variant of the maltose-binding protein. Sci. Rep. 9, 20396 (2019).
Article CAS PubMed PubMed Central Google Scholar
Cordeiro, T. N. et al. Small-angle scattering studies of intrinsically disordered proteins and their complexes. Curr. Opin. Struct. Biol. 42, 15–23 (2017).
Article CAS PubMed Google Scholar
Westerlund, A. M. & Delemotte, L. Effect of Ca2+ on the promiscuous target-protein binding of calmodulin. PLoS Comput Biol. 14, e1006072 (2018).
Article PubMed PubMed Central CAS Google Scholar
Kumar, V. et al. Structural basis for the interaction of unstructured neuron specific substrates neuromodulin and neurogranin with Calmodulin. Sci. Rep. 3, 1392 (2013).
Article PubMed PubMed Central CAS Google Scholar
Della Rocca, G. J. et al. Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptors. Convergence of Gi- and Gq-mediated pathways on calcium/calmodulin, Pyk2, and Src kinase. J. Biol. Chem. 272, 19125–19132 (1997).
Article CAS PubMed Google Scholar
Nicodemo, A. A. et al. Pyk2 uncouples metabotropic glutamate receptor G protein signaling but facilitates ERK1/2 activation. Mol. Brain 3, 4 (2010).
Article PubMed PubMed Central CAS Google Scholar
Fuxreiter, M. Classifying the binding modes of disordered proteins. Int. J. Mol. Sci. 21, 8615 (2020).
Wang, X. & Putkey, J. A. PEP-19 modulates calcium binding to calmodulin by electrostatic steering. Nat. Commun. 7, 13583 (2016).
Article CAS PubMed PubMed Central Google Scholar
Yamauchi, E., Nakatsu, T., Matsubara, M., Kato, H. & Taniguchi, H. Crystal structure of a MARCKS peptide containing the calmodulin-binding domain in complex with Ca2+-calmodulin. Nat. Struct. Biol. 10, 226–231 (2003).
Article CAS PubMed Google Scholar
Koster, S., Pavkov-Keller, T., Kuhlbrandt, W. & Yildiz, O. Structure of human Na+/H+ exchanger NHE1 regulatory region in complex with calmodulin and Ca₂. J. Biol. Chem. 286, 40954–40961 (2011).
Article PubMed PubMed Central CAS Google Scholar
Malini Nagulapalli, G. P. et al. Recognition pliability is coupled to structural heterogeneity: a calmodulin intrinsically disordered binding region complex. Structure 20, 522–533 (2012).
Article PubMed CAS Google Scholar
Danielsson, J. et al. The intrinsically disordered RNR inhibitor Sml1 is a dynamic dimer. Biochemistry 47, 13428–13437 (2008).
Article CAS PubMed Google Scholar
Wang, W. et al. A soluble alpha-synuclein construct forms a dynamic tetramer. Proc. Natl Acad. Sci. USA 108, 17797–17802 (2011).
Article CAS PubMed PubMed Central Google Scholar
Lah, J. S. M. et al. Energetics of structural transitions of the addiction antitoxin MazE: is a programmed bacterial cell death dependent on the intrinsically flexible nature of the antitoxins? J. Biol. Chem. 280, 17397–17407 (2005).
Article CAS PubMed Google Scholar
Alonso, L. G. A. G.-A. et al. High-risk (HPV16) human papillomavirus E7 oncoprotein is highly stable and extended, with conformational transitions that could explain its multiple cellular binding partners. Biochemistry 41, 10510–10518 (2002).
Article CAS PubMed Google Scholar
Frost, L. et al. The dimerization state of the mammalian high mobility group protein AT-hook 2 (HMGA2). PLoS ONE 10, e0130478 (2015).
Article PubMed PubMed Central CAS Google Scholar
Ansari, M. Z., Alom, S. E. & Swaminathan, R. Ordered structure induced in human c-Myc PEST region upon forming a disulphide bonded dimer. J. Chem. Sci. 133, 26 (2021).
Rahman, S. K. O. H. & Chen, Y. W. Frameshift PQBP-1 mutants K192Sfs*7 and R153Sfs*41 implicated in X-linked intellectual disability form stable dimers. J. Struct. Biol. 206, 305–313 (2019).
Article CAS PubMed Google Scholar
Sigalov, A., Aivazian, D. & Stern, L. Homooligomerization of the cytoplasmic domain of the T cell receptor zeta chain and of other proteins containing the immunoreceptor tyrosine-based activation motif. Biochemistry 43, 2049–2061 (2004).
Article CAS PubMed Google Scholar
Dasgupta, I., Sanglas, L., Enghild, J. J. & Lindberg, I. The neuroendocrine protein 7B2 is intrinsically disordered. Biochemistry 51, 7456–7464 (2012).
Article CAS PubMed Google Scholar
Lanza, D. C. et al. Human FEZ1 has characteristics of a natively unfolded protein and dimerizes in solution. Proteins 74, 104–121 (2009).
Article CAS PubMed Google Scholar
Pieprzyk, J., Zbela, A., Jakob, M., Ozyhar, A. & Orlowski, M. Homodimerization propensity of the intrinsically disordered N-terminal domain of Ultraspiracle from Aedes aegypti. Biochim. Biophys. Acta 1844, 1153–1166 (2014).
Article CAS PubMed Google Scholar
Raja, S., Avraham, S. & Avraham, H. Tyrosine phosphorylation of the novel protein-tyrosine kinase RAFTK during an early phase of platelet activation by an integrin glycoprotein IIb-IIIa-independent mechanism. J. Biol. Chem. 272, 10941–10947 (1997).
Article CAS PubMed Google Scholar
Sjogaard-Frich, L. M. et al. Dynamic Na(+)/H(+) exchanger 1 (NHE1)-calmodulin complexes of varying stoichiometry and structure regulate Ca(2+)-dependent NHE1 activation. Elife 10, e60889 (2021).
Li, Z., Zhang, Y., Hedman, A. C., Ames, J. B. & Sacks, D. B. Calmodulin lobes facilitate dimerization and activation of estrogen receptor-alpha. J. Biol. Chem. 292, 4614–4622 (2017).
Article CAS PubMed PubMed Central Google Scholar
Shahul Hameed, U. F. et al. H-NS uses an autoinhibitory conformational switch for environment-controlled gene silencing. Nucleic Acids Res. 47, 2666–2680 (2019).
Article PubMed CAS Google Scholar
Diaz Galicia, M. E., Aldehaiman, A., Hong, S., Arold, S. T. & Grunberg, R. Methods for the recombinant expression of active tyrosine kinase domains: guidelines and pitfalls. Methods Enzymol. 621, 131–152 (2019).
Article PubMed CAS Google Scholar
Wiedemann, C., Bellstedt, P. & Gorlach, M. CAPITO–a web server-based analysis and plotting tool for circular dichroism data. Bioinformatics 29, 1750–1757 (2013).
Article CAS PubMed Google Scholar
Zhao H, Brautigam CA, Ghirlando R, Schuck P. Overview of current methods in sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. Curr. Protoc. Protein Sci. Chapter 20, Unit20.12 (2013).
Vistica, J. et al. Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition. Anal. Biochem. 326, 234–256 (2004).
Article CAS PubMed Google Scholar
Williamson, M. P. Using chemical shift perturbation to characterise ligand binding. Prog. Nucl. Magn. Reson Spectrosc. 73, 1–16 (2013).
Article CAS PubMed Google Scholar
Lee, W., Tonelli, M. & Markley, J. L. NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 31, 1325–1327 (2015).
Article PubMed Google Scholar
Qian, Y., Shen, M., Amoureux, J. P., Noda, I. & Hu, B. The dependence of signal-to-noise ratio on number of scans in covariance spectroscopy. Solid State Nucl. Magn. Reson. 59-60, 31–33 (2014).
Article CAS PubMed Google Scholar
Evans, J. N. S. Biomolecular NMR Spectroscopy (Oxford University Press, 1995).
Guntert, P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278, 353–378 (2004).
CAS PubMed Google Scholar
Shen, Y. & Bax, A. Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks. J. Biomol. NMR 56, 227–241 (2013).
Article CAS PubMed PubMed Central Google Scholar
Nederveen, A. J. et al. RECOORD: a recalculated coordinate database of 500+ proteins from the PDB using restraints from the BioMagResBank. Proteins 59, 662–672 (2005).
Article CAS PubMed Google Scholar
Rost, B. PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol. 266, 525–539 (1996).
Article CAS PubMed Google Scholar
McGuffin, L. J., Bryson, K. & Jones, D. T. The PSIPRED protein structure prediction server. Bioinformatics 16, 404–405 (2000).
Article CAS PubMed Google Scholar
Cuff, J. A., Clamp, M. E., Siddiqui, A. S., Finlay, M. & Barton, G. J. JPred: a consensus secondary structure prediction server. Bioinformatics 14, 892–893 (1998).
Article CAS PubMed Google Scholar
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Article CAS PubMed PubMed Central Google Scholar
Lupas, A., Van Dyke, M. & Stock, J. Predicting coiled coils from protein sequences. Science 252, 1162–1164 (1991).
Article CAS PubMed Google Scholar
Simm, D., Hatje, K. & Kollmar, M. Waggawagga: comparative visualization of coiled-coil predictions and detection of stable single alpha-helices (SAH domains). Bioinformatics 31, 767–769 (2015).
Article CAS PubMed Google Scholar
Mruk, K., Farley, B. M., Ritacco, A. W. & Kobertz, W. R. Calmodulation meta-analysis: predicting calmodulin binding via canonical motif clustering. J. Gen. Physiol. 144, 105–114 (2014).
Article CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

We acknowledge SOLEIL for providing synchrotron radiation facilities (proposals nr. 20181104 and 20180576). We would also like to thank J. Perez and A. Thureau for assistance in using the beamline SWING. We thank the KAUST Bioscience, and Imaging and Characterisation core labs for their assistance. We thank T.M.D. Besong and O. Bakr from the Functional Nanomaterials Lab at KAUST for their help with the AUC measurement and analysis. We thank D. Renn and M. Rue** (KAUST Catalysis Center) for the access to the CD instrument. We also thank U.S. Hameed for discussions on the manuscript and R. Naser for help with NMR data. This publication is based on the work supported by the King Abdullah University of Science and Technology (KAUST) Office of Sponsored Research (OSR) under the Award Number URF/1/2602-01-01. The work in JAG’s lab was supported by grants from Agence Nationale de la Recherche (ANR-19-CE16-0020) and Fondation pour la Recherche Médicale (FRM, EQU201903007844). P.B. acknowledges support from the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05.

Author information

These authors contributed equally: Tiago Mendes, Philippe Barthe.

Authors and Affiliations

Computational Bioscience Research Center (CBRC), King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia
Afaque A. Momin, SeungBeom Hong, Piao Yu & Stefan T. Arold
Bioscience Program, Division of Biological and Environmental Science and Engineering (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia
Afaque A. Momin, SeungBeom Hong, Piao Yu, Mariusz Jaremko, Łukasz Jaremko & Stefan T. Arold
Inserm UMR-S 1270, Sorbonne Université, Faculty of Sciences and Engineering, Institut du Fer à Moulin, 75005, Paris, France
Tiago Mendes, Camille Faure, Gress Kadaré & Jean-Antoine Girault
Centre de Biologie Structurale (CBS), University Montpellier, INSERM U1054, CNRS UMR 5048, 34090, Montpellier, France
Philippe Barthe & Stefan T. Arold

Authors

Afaque A. Momin
View author publications
You can also search for this author in PubMed Google Scholar
Tiago Mendes
View author publications
You can also search for this author in PubMed Google Scholar
Philippe Barthe
View author publications
You can also search for this author in PubMed Google Scholar
Camille Faure
View author publications
You can also search for this author in PubMed Google Scholar
SeungBeom Hong
View author publications
You can also search for this author in PubMed Google Scholar
Piao Yu
View author publications
You can also search for this author in PubMed Google Scholar
Gress Kadaré
View author publications
You can also search for this author in PubMed Google Scholar
Mariusz Jaremko
View author publications
You can also search for this author in PubMed Google Scholar
Jean-Antoine Girault
View author publications
You can also search for this author in PubMed Google Scholar
Łukasz Jaremko
View author publications
You can also search for this author in PubMed Google Scholar
Stefan T. Arold
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

Protein cloning, expression and purification: A.A.M. and S.H. Biochemical and biophysical analysis: A.A.M., S.H., P.Y. NMR analysis: A.A.M., P.B., M.J., Ł.J. SAXS analysis: A.A.M., S.T.A. Cell biology: T.M., C.F., G.K., J.A.G. Project design and supervision: S.T.A., J.A.G., P.B., M.J., Ł.J. Manuscript writing: A.A.M., S.T.A., T.M., J.A.G., Ł.J. All authors read and approved the manuscript.

Corresponding author

Correspondence to Stefan T. Arold.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary handling editors: Krishnananda Chattopadhyay, Anam Akhtar and Christina Karlsson Rosenthal. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Peer Review File

Supplementary Information

Description of Additional Supplementary Files

Supplementary Data

Reporting Summary

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Momin, A.A., Mendes, T., Barthe, P. et al. PYK2 senses calcium through a disordered dimerization and calmodulin-binding element. Commun Biol 5, 800 (2022). https://doi.org/10.1038/s42003-022-03760-8

Download citation

Received: 14 December 2021
Accepted: 22 July 2022
Published: 09 August 2022
DOI: https://doi.org/10.1038/s42003-022-03760-8
Springer Nature Limited

PYK2 senses calcium through a disordered dimerization and calmodulin-binding element

Abstract

Similar content being viewed by others

Introduction

PYK2-KFL binds to CaM in a process involving more than one linear peptide association

PYK2 control through the fuzzy dimeric CaM-binding element

Discussion

Methods

Cloning

Expression and purification

Circular dichroism (CD)

Analytical size-exclusion chromatography (SEC)

Analytical ultracentrifugation (AUC)

Microscale thermophoresis (MST)

Fluorescence anisotropy

Differential-scanning fluorimetry

Isothermal titration calorimetry (ITC)

Nuclear magnetic resonance (NMR)

Visible immunoprecipitation (VIP)

Cell culture

Pulldown assay and immunoblot

Bioinformatics

Statistics and reproducibility

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing interests

Peer review

Peer review information

Additional information

Supplementary information

Rights and permissions

About this article

Cite this article

Share this article

Search

Navigation