TY - JOUR
T1 - Ligand-induced CaMKIIα hub Trp403 flip, hub domain stacking, and modulation of kinase activity
AU - Narayanan, Dilip
AU - Larsen, Anne Sofie G
AU - Gauger, Stine Juul
AU - Adafia, Ruth
AU - Hammershøi, Rikke Bartschick
AU - Hamborg, Louise
AU - Bruus-Jensen, Jesper
AU - Griem-Krey, Nane
AU - Gee, Christine L
AU - Frølund, Bente
AU - Stratton, Margaret M
AU - Kuriyan, John
AU - Kastrup, Jette Sandholm
AU - Langkilde, Annette E
AU - Wellendorph, Petrine
AU - Solbak, Sara M Ø
N1 - © 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.
PY - 2024
Y1 - 2024
N2 - γ-Hydroxybutyric acid (GHB) analogs are small molecules that bind competitively to a specific cavity in the oligomeric CaMKIIα hub domain. Binding affects conformation and stability of the hub domain, which may explain the neuroprotective action of some of these compounds. Here, we describe molecular details of interaction of the larger-type GHB analog 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid (PIPA). Like smaller-type analogs, PIPA binding to the CaMKIIα hub domain promoted thermal stability. PIPA additionally modulated CaMKIIα activity under sub-maximal CaM concentrations and ultimately led to reduced substrate phosphorylation. A high-resolution X-ray crystal structure of a stabilized CaMKIIα (6x mutant) hub construct revealed details of the binding mode of PIPA, which involved outward placement of tryptophan 403 (Trp403), a central residue in a flexible loop close to the upper hub cavity. Small-angle X-ray scattering (SAXS) solution structures and mass photometry of the CaMKIIα wild-type hub domain in the presence of PIPA revealed a high degree of ordered self-association (stacks of CaMKIIα hub domains). This stacking neither occurred with the smaller compound 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), nor when Trp403 was replaced with leucine (W403L). Additionally, CaMKIIα W403L hub was stabilized to a larger extent by PIPA compared to CaMKIIα hub wild type, indicating that loop flexibility is important for holoenzyme stability. Thus, we propose that ligand-induced outward placement of Trp403 by PIPA, which promotes an unforeseen mechanism of hub domain stacking, may be involved in the observed reduction in CaMKIIα kinase activity. Altogether, this sheds new light on allosteric regulation of CaMKIIα activity via the hub domain.
AB - γ-Hydroxybutyric acid (GHB) analogs are small molecules that bind competitively to a specific cavity in the oligomeric CaMKIIα hub domain. Binding affects conformation and stability of the hub domain, which may explain the neuroprotective action of some of these compounds. Here, we describe molecular details of interaction of the larger-type GHB analog 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid (PIPA). Like smaller-type analogs, PIPA binding to the CaMKIIα hub domain promoted thermal stability. PIPA additionally modulated CaMKIIα activity under sub-maximal CaM concentrations and ultimately led to reduced substrate phosphorylation. A high-resolution X-ray crystal structure of a stabilized CaMKIIα (6x mutant) hub construct revealed details of the binding mode of PIPA, which involved outward placement of tryptophan 403 (Trp403), a central residue in a flexible loop close to the upper hub cavity. Small-angle X-ray scattering (SAXS) solution structures and mass photometry of the CaMKIIα wild-type hub domain in the presence of PIPA revealed a high degree of ordered self-association (stacks of CaMKIIα hub domains). This stacking neither occurred with the smaller compound 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), nor when Trp403 was replaced with leucine (W403L). Additionally, CaMKIIα W403L hub was stabilized to a larger extent by PIPA compared to CaMKIIα hub wild type, indicating that loop flexibility is important for holoenzyme stability. Thus, we propose that ligand-induced outward placement of Trp403 by PIPA, which promotes an unforeseen mechanism of hub domain stacking, may be involved in the observed reduction in CaMKIIα kinase activity. Altogether, this sheds new light on allosteric regulation of CaMKIIα activity via the hub domain.
KW - Calcium-Calmodulin-Dependent Protein Kinase Type 2/chemistry
KW - Crystallography, X-Ray
KW - Humans
KW - Protein Domains
KW - Ligands
KW - Models, Molecular
KW - Scattering, Small Angle
KW - Tryptophan/chemistry
KW - Pyridazines/chemistry
KW - Phosphorylation
U2 - 10.1002/pro.5152
DO - 10.1002/pro.5152
M3 - Journal article
C2 - 39275999
VL - 33
JO - Protein Science
JF - Protein Science
SN - 0961-8368
IS - 10
M1 - e5152
ER -