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Munc13-like skMLCK variants cannot mimic the unique calmodulin binding mode of Munc13 as evidenced by chemical cross-linking and mass spectrometry.

Herbst S, Maucher D, Schneider M, Ihling CH, Jahn O, Sinz A - PLoS ONE (2013)

Bottom Line: This unusual binding mode is thought to be related to the presence of an additional hydrophobic anchor residue at position 26 of the CaM binding motif of Munc13-1, resulting in a novel 1-5-8-26 motif.Apparently, additional features apart from the spacing of the hydrophobic anchor residues are required to define the functional 1-5-8-26 motif of Munc13-1.We conclude that Munc13 proteins display a unique CaM binding behavior to fulfill their role as efficient presynaptic calcium sensors over broad range of Ca(2+) concentrations.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.

ABSTRACT
Among the neuronal binding partners of calmodulin (CaM) are Munc13 proteins as essential presynaptic regulators that play a key role in synaptic vesicle priming and are crucial for presynaptic short-term plasticity. Recent NMR structural investigations of a CaM/Munc13-1 peptide complex have revealed an extended structure, which contrasts the compact structures of most classical CaM/target complexes. This unusual binding mode is thought to be related to the presence of an additional hydrophobic anchor residue at position 26 of the CaM binding motif of Munc13-1, resulting in a novel 1-5-8-26 motif. Here, we addressed the question whether the 1-5-8-26 CaM binding motif is a Munc13-related feature or whether it can be induced in other CaM targets by altering the motif's core residues. For this purpose, we chose skeletal muscle myosin light chain kinase (skMLCK) with a classical 1-5-8-14 CaM binding motif and constructed three skMLCK peptide variants mimicking Munc13-1, in which the hydrophobic anchor amino acid at position 14 was moved to position 26. Chemical cross-linking between CaM and skMLCK peptide variants combined with high-resolution mass spectrometry yielded insights into the peptides' binding modes. This structural comparison together with complementary binding data from surface plasmon resonance experiments revealed that skMLCK variants with an artificial 1-5-8-26 motif cannot mimic CaM binding of Munc13-1. Apparently, additional features apart from the spacing of the hydrophobic anchor residues are required to define the functional 1-5-8-26 motif of Munc13-1. We conclude that Munc13 proteins display a unique CaM binding behavior to fulfill their role as efficient presynaptic calcium sensors over broad range of Ca(2+) concentrations.

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SDS-PAGE of cross-linking reaction mixtures between CaM and skMLCK peptides.(A) Cross-linking reactions between CaM and skMLCK peptide (50-fold molar excess of BS2G, Ca2+ concentrations: 0 to 50 mM). As a control, CaM was diluted in water without adding buffer, EGTA, or Ca2+; please note the different Ca2+-loaded states of CaM (lane 10). (B) Cross-linking reactions between CaM and the skMLCK F19E/L31W peptide, conducted at 50-fold molar excess of SBC. Lanes 1–3: 1 mM Ca2+; lanes 5–7: 30 nM Ca2+. M: protein marker.
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pone-0075119-g002: SDS-PAGE of cross-linking reaction mixtures between CaM and skMLCK peptides.(A) Cross-linking reactions between CaM and skMLCK peptide (50-fold molar excess of BS2G, Ca2+ concentrations: 0 to 50 mM). As a control, CaM was diluted in water without adding buffer, EGTA, or Ca2+; please note the different Ca2+-loaded states of CaM (lane 10). (B) Cross-linking reactions between CaM and the skMLCK F19E/L31W peptide, conducted at 50-fold molar excess of SBC. Lanes 1–3: 1 mM Ca2+; lanes 5–7: 30 nM Ca2+. M: protein marker.

Mentions: Before conducting the cross-linking experiments for 3D-structural analysis of CaM/peptide complexes, we performed Ca2+ titration experiments to investigate the influence of Ca2+ on CaM/skMLCK peptide complex formation. Figure 2A shows an SDS gel of cross-linking reactions with Ca2+ concentrations ranging from 0 to 50 mM. It is readily visible that 1∶1 complexes between CaM and the skMLCK peptide are created with the cross-linker BS2G both at low and higher Ca2+ concentrations; yet, the CaM/skMLCK peptide complexes created at resting, i.e., nanomolar, Ca2+ concentrations seem to be different from those created at higher Ca2+ concentrations [25]. A dramatic change in the electrophoretic mobility of CaM is detected at Ca2+ concentrations between 100 nM and 100 µM due to a conformational change in CaM upon calcium binding (KD (C-terminal lobe of CaM): 2 · 10−7 M; KD (N-terminal lobe of CaM): 2 · 10−6 M [26]). Moreover, at higher Ca2+ concentrations (>100 µM) there are several bands visible corresponding to different cross-linked species (both intramolecular cross-links in CaM as well as intermolecular cross-links between CaM and the peptide) exhibiting slightly different electrophoretic mobilities. Subsequent cross-linking experiments were conducted at two different Ca2+ concentrations, 1 mM and 30 nM (Figure 2B and Figure S2 in File S1), in order to investigate the binding behavior of skMLCK peptides to CaM both in the low and the high calcium range.


Munc13-like skMLCK variants cannot mimic the unique calmodulin binding mode of Munc13 as evidenced by chemical cross-linking and mass spectrometry.

Herbst S, Maucher D, Schneider M, Ihling CH, Jahn O, Sinz A - PLoS ONE (2013)

SDS-PAGE of cross-linking reaction mixtures between CaM and skMLCK peptides.(A) Cross-linking reactions between CaM and skMLCK peptide (50-fold molar excess of BS2G, Ca2+ concentrations: 0 to 50 mM). As a control, CaM was diluted in water without adding buffer, EGTA, or Ca2+; please note the different Ca2+-loaded states of CaM (lane 10). (B) Cross-linking reactions between CaM and the skMLCK F19E/L31W peptide, conducted at 50-fold molar excess of SBC. Lanes 1–3: 1 mM Ca2+; lanes 5–7: 30 nM Ca2+. M: protein marker.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3794949&req=5

pone-0075119-g002: SDS-PAGE of cross-linking reaction mixtures between CaM and skMLCK peptides.(A) Cross-linking reactions between CaM and skMLCK peptide (50-fold molar excess of BS2G, Ca2+ concentrations: 0 to 50 mM). As a control, CaM was diluted in water without adding buffer, EGTA, or Ca2+; please note the different Ca2+-loaded states of CaM (lane 10). (B) Cross-linking reactions between CaM and the skMLCK F19E/L31W peptide, conducted at 50-fold molar excess of SBC. Lanes 1–3: 1 mM Ca2+; lanes 5–7: 30 nM Ca2+. M: protein marker.
Mentions: Before conducting the cross-linking experiments for 3D-structural analysis of CaM/peptide complexes, we performed Ca2+ titration experiments to investigate the influence of Ca2+ on CaM/skMLCK peptide complex formation. Figure 2A shows an SDS gel of cross-linking reactions with Ca2+ concentrations ranging from 0 to 50 mM. It is readily visible that 1∶1 complexes between CaM and the skMLCK peptide are created with the cross-linker BS2G both at low and higher Ca2+ concentrations; yet, the CaM/skMLCK peptide complexes created at resting, i.e., nanomolar, Ca2+ concentrations seem to be different from those created at higher Ca2+ concentrations [25]. A dramatic change in the electrophoretic mobility of CaM is detected at Ca2+ concentrations between 100 nM and 100 µM due to a conformational change in CaM upon calcium binding (KD (C-terminal lobe of CaM): 2 · 10−7 M; KD (N-terminal lobe of CaM): 2 · 10−6 M [26]). Moreover, at higher Ca2+ concentrations (>100 µM) there are several bands visible corresponding to different cross-linked species (both intramolecular cross-links in CaM as well as intermolecular cross-links between CaM and the peptide) exhibiting slightly different electrophoretic mobilities. Subsequent cross-linking experiments were conducted at two different Ca2+ concentrations, 1 mM and 30 nM (Figure 2B and Figure S2 in File S1), in order to investigate the binding behavior of skMLCK peptides to CaM both in the low and the high calcium range.

Bottom Line: This unusual binding mode is thought to be related to the presence of an additional hydrophobic anchor residue at position 26 of the CaM binding motif of Munc13-1, resulting in a novel 1-5-8-26 motif.Apparently, additional features apart from the spacing of the hydrophobic anchor residues are required to define the functional 1-5-8-26 motif of Munc13-1.We conclude that Munc13 proteins display a unique CaM binding behavior to fulfill their role as efficient presynaptic calcium sensors over broad range of Ca(2+) concentrations.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.

ABSTRACT
Among the neuronal binding partners of calmodulin (CaM) are Munc13 proteins as essential presynaptic regulators that play a key role in synaptic vesicle priming and are crucial for presynaptic short-term plasticity. Recent NMR structural investigations of a CaM/Munc13-1 peptide complex have revealed an extended structure, which contrasts the compact structures of most classical CaM/target complexes. This unusual binding mode is thought to be related to the presence of an additional hydrophobic anchor residue at position 26 of the CaM binding motif of Munc13-1, resulting in a novel 1-5-8-26 motif. Here, we addressed the question whether the 1-5-8-26 CaM binding motif is a Munc13-related feature or whether it can be induced in other CaM targets by altering the motif's core residues. For this purpose, we chose skeletal muscle myosin light chain kinase (skMLCK) with a classical 1-5-8-14 CaM binding motif and constructed three skMLCK peptide variants mimicking Munc13-1, in which the hydrophobic anchor amino acid at position 14 was moved to position 26. Chemical cross-linking between CaM and skMLCK peptide variants combined with high-resolution mass spectrometry yielded insights into the peptides' binding modes. This structural comparison together with complementary binding data from surface plasmon resonance experiments revealed that skMLCK variants with an artificial 1-5-8-26 motif cannot mimic CaM binding of Munc13-1. Apparently, additional features apart from the spacing of the hydrophobic anchor residues are required to define the functional 1-5-8-26 motif of Munc13-1. We conclude that Munc13 proteins display a unique CaM binding behavior to fulfill their role as efficient presynaptic calcium sensors over broad range of Ca(2+) concentrations.

Show MeSH
Related in: MedlinePlus