Determination of residue specificity in the EF-hand of troponin C for Ca2+ coordination, by genetic engineering.

Babu, A.; Hong, Seonki; Ryu, Yongju; Gulati, Jagdish

doi:10.1016/s0021-9258(19)49558-6

Cited by 83 publications

(37 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The most likely interpretation of the functional effects of the N283E, D346E, and D408E mutations is that the presence of a larger amino acid (Glu) may have disrupted the tertiary structure at the cation binding site, and perhaps even prevented cation binding. In this regard, substitution of Glu for Asp in position 3 of a divalent cation site in troponin C yielded functionally inactive protein with concomitantly diminished cation binding capacity (5,53).…”

Section: Implications Regarding Divalent Cation Sitesmentioning

confidence: 99%

“…From the studies of calmodulin and troponin C, an EF-hand is a loop structure in which acidic amino acids (Asp or Glu), together with other oxygen containing amino acids (at positions 1, 3, 5, 7, 9, 12), provide 6 or 7 coordination sites for a divalent cation (68). Conservative mutations, or alanine substitutions, at positions 1, 3, and 12 within troponin C EF-hands emphasize that those residues are critical for both function and cation binding (5,53). In another study, conservative mutations at position 9 of the EF-hand of the Escherichia Coli D-glucose and D-galactose receptor caused a change in both cation selectivity and affinity (16).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Mutation of putative divalent cation sites in the alpha 4 subunit of the integrin VLA-4: distinct effects on adhesion to CS1/fibronectin, VCAM-1, and invasin.

Masumoto

Hemler

1993

The Journal of Cell Biology

109

View full text Add to dashboard Cite

Abstract. To investigate the functional significance of putative integrin divalent cation binding sites, several mutated 0/4 subunit cDNAs were constructed. Mutants contained the conservative substitution of Glu for Asp or Asn at the third position in each of three putative divalent cation sites. Transfection of wild-type or mutated ~ into K562 cells yielded comparable expression levels and immunoprecipitation profiles. However, for all three ~ mutants, adhesion to CS1/fibronectin was greatly diminished in either the presence or absence of the stimulatory anti-ill mAb TS2/16. Constitutive adhesion to vascular cell adhesion molecule (VCAM) 1 was also diminished but, unlike CS1 adhesion, was restored upon TS2/16 stimulation. In contrast, adhesion to the bacterial protein invasin was minimally affected by any of the three mutations. For each of the mutants, the order of preference for divalent cations was unchanged compared to wild-type O/4, on CS1/fibronectin (Mn 2÷ > Mg 2÷ > Ca2÷), on VCAM-1 (Mn 2÷ > Mg 2÷ = Ca 2÷) and on invasin (Mg 2÷ = Ca2÷). However for the three mutants, the efficiency of divalent cation utilization was decreased. On VCAM-1, 68-108/zM Mn 2÷ was required to support halfmaximal adhesion for the mutants compared with 14-18/~M for wild-type 0/4. These results indicate (a) that three different ligands for VLA-4 show widely differing sensitivities to mutations within putative divalent cation sites, and (b) each of the three putative divalent cation sites in ~ have comparable functional importance with respect to both divalent cation usage and cell adhesion.

show abstract

Section: Implications Regarding Divalent Cation Sitesmentioning

confidence: 99%

mentioning

confidence: 99%

Mutation of putative divalent cation sites in the alpha 4 subunit of the integrin VLA-4: distinct effects on adhesion to CS1/fibronectin, VCAM-1, and invasin.

Masumoto

Hemler

1993

The Journal of Cell Biology

109

View full text Add to dashboard Cite

show abstract

“…In evolutionary aspects, not surprisingly, the acidic residues on the CaM loop; Asp-93, Asp-95, and Glu-104, are highly conserved among virtually all EF-hand Ca 21 binding motifs (34). Point mutations in these residues of the EF-hand motif basically eliminate the Ca 21 binding ability unless extremely high Ca 21 concentrations are explored (35)(36)(37)(38)(39).…”

Section: Discussionmentioning

confidence: 99%

Protein Grabs a Ligand by Extending Anchor Residues: Molecular Simulation for Ca2+ Binding to Calmodulin Loop

Kobayashi¹,

Takada

2006

Biophysical Journal

View full text Add to dashboard Cite

The structural difference in proteins between unbound and bound forms directly suggests the importance of the conformational plasticity of proteins. However, pathways that connect two-end structures and how they are coupled to the binding reaction are not well understood at atomic resolution. Here, we analyzed the free-energy landscape, explicitly taking into account coupling between binding and conformational change by performing atomistic molecular dynamics simulations for Ca2+ binding to a calmodulin loop. Using the AMBER force field with explicit water solvent, we conducted umbrella sampling for the free-energy surface and steered molecular dynamics for the pathway search. We found that, at an early stage of binding, some key residue side chains extend their "arms" to catch Ca2+ and, after catching, they carry the Ca2+ to the center of the binding pocket. This grabbing motion resulted in smooth and stepwise exchange in coordination partners of Ca2+ from water oxygen to atoms in the calmodulin loop. The key residue that first caught the ion was one of the two acidic residues, which are highly conserved. In the pathway simulations, different pathways were observed between binding and dissociation reactions: The former was more diverse than the latter.

show abstract

“…The isolation of cTnC WT/G34S was performed following the method described by Babu et al (1992) [54]. cTnI WT/D127Y were purified according to Reiffert et al (1999) [55].…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

De Novo Missense Mutations in TNNC1 and TNNI3 Causing Severe Infantile Cardiomyopathy Affect Myofilament Structure and Function and Are Modulated by Troponin Targeting Agents

Hassoun

Budde

Mannherz

et al. 2021

IJMS

View full text Add to dashboard Cite

Rare pediatric non-compaction and restrictive cardiomyopathy are usually associated with a rapid and severe disease progression. While the non-compaction phenotype is characterized by structural defects and is correlated with systolic dysfunction, the restrictive phenotype exhibits diastolic dysfunction. The molecular mechanisms are poorly understood. Target genes encode among others, the cardiac troponin subunits forming the main regulatory protein complex of the thin filament for muscle contraction. Here, we compare the molecular effects of two infantile de novo point mutations in TNNC1 (p.cTnC-G34S) and TNNI3 (p.cTnI-D127Y) leading to severe non-compaction and restrictive phenotypes, respectively. We used skinned cardiomyocytes, skinned fibers, and reconstituted thin filaments to measure the impact of the mutations on contractile function. We investigated the interaction of these troponin variants with actin and their inter-subunit interactions, as well as the structural integrity of reconstituted thin filaments. Both mutations exhibited similar functional and structural impairments, though the patients developed different phenotypes. Furthermore, the protein quality control system was affected, as shown for TnC-G34S using patient’s myocardial tissue samples. The two troponin targeting agents levosimendan and green tea extract (-)-epigallocatechin-3-gallate (EGCg) stabilized the structural integrity of reconstituted thin filaments and ameliorated contractile function in vitro in some, but not all, aspects to a similar degree for both mutations.

show abstract

Determination of residue specificity in the EF-hand of troponin C for Ca2+ coordination, by genetic engineering.

Cited by 83 publications

References 29 publications

Mutation of putative divalent cation sites in the alpha 4 subunit of the integrin VLA-4: distinct effects on adhesion to CS1/fibronectin, VCAM-1, and invasin.

Mutation of putative divalent cation sites in the alpha 4 subunit of the integrin VLA-4: distinct effects on adhesion to CS1/fibronectin, VCAM-1, and invasin.

Protein Grabs a Ligand by Extending Anchor Residues: Molecular Simulation for Ca2+ Binding to Calmodulin Loop

De Novo Missense Mutations in TNNC1 and TNNI3 Causing Severe Infantile Cardiomyopathy Affect Myofilament Structure and Function and Are Modulated by Troponin Targeting Agents

Contact Info

Product

Resources

About