Source 1primary paper2008Biochemistry
Toolkit/EF-III metal-binding site of CIB1
EF-III metal-binding site of CIB1
Also known as: EF-III
Taxonomy: Mechanism Branch / Component. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
The EF-III metal-binding site of calcium- and integrin-binding protein 1 (CIB1) is an EF-hand domain characterized as a mixed Mg2+/Ca2+-binding site. Mutational analysis of its metal-coordinating loop indicates that EF-III modulates CIB1 affinity for the integrin alphaIIb domain and tunes metal sensitivity into a range relevant to intracellular Ca2+ sensing.
Usefulness & Problems
Why this is useful
EF-III is useful as a defined metal-responsive regulatory element within CIB1 for studying how EF-hand loop chemistry controls divalent cation selectivity and ligand binding. The available mutational data support its use in dissecting coupling between Mg2+/Ca2+ coordination and alphaIIb recognition.
Problem solved
This domain helps address the problem of identifying how a single EF-hand site can support mixed Mg2+/Ca2+ binding while influencing protein-ligand interactions. The cited work specifically links EF-III loop composition to altered metal affinity and to changes in CIB1 binding to the integrin alphaIIb domain.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Component: A low-level protein part used inside a larger architecture that realizes a mechanism.
Mechanisms
divalent metal ion bindingmetal-dependent modulation of protein-ligand bindingmixed mg2+/ca2+ coordinationTechniques
Structural CharacterizationTarget processes
recombinationImplementation Constraints
Practical use of EF-III requires preserving its metal-coordinating loop context within CIB1, because the reported functional effects derive from site-directed substitutions in that loop. The relevant readouts in the cited study were Mg2+ and Ca2+ binding at EF-III and binding to the integrin alphaIIb domain; no additional expression, delivery, or construct-format details are provided in the supplied evidence.
The evidence is limited to one cited study focused on CIB1 mutational analysis and alphaIIb-domain binding, with no broader validation across other ligands, organisms, or applications. Some mutations uncouple metal binding from detectable ligand interaction, as the D127E variant increased Mg2+ and Ca2+ affinity at EF-III but had alphaIIb-domain binding too weak to observe.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
CIB1 EF-III is a mixed Mg2+/Ca2+-binding site.
EF-III is a mixed Mg2+/Ca2+-binding site
CIB1 EF-IV is a higher-affinity Ca2+-specific site.
EF-IV is a higher-affinity Ca2+-specific site
Replacement of the ancestral Glu with Asp at the -Z position of EF-III increased CIB1 affinity for alphaIIb and shifted Ca2+ affinity into a range compatible with intracellular Ca2+ sensing.
upon replacement of the ancestral Glu with Asp at the -Z position of EF-III, CIB1 gained affinity for alphaIIb, and the Ca2+ affinity of CIB1 shifted into a range where the protein is able to act as an intracellular Ca2+ sensor
The CIB1 D127E mutant has increased Mg2+ and Ca2+ affinity at EF-III, but alphaIIb-domain binding is too weak to observe.
A D127E mutant had increased Mg2+ and Ca2+ affinity at EF-III, but unexpectedly, the affinity for the alphaIIb domain was too low for binding to be observed.
The CIB1 D127N mutant has reduced Mg2+ and Ca2+ affinity at EF-III but retains affinity for the alphaIIb domain.
A D127N mutant had reduced Mg2+ and Ca2+ affinity at EF-III but retained affinity for the alphaIIb domain.
The CIB1 E172D mutant shows weak Mg2+ binding at EF-IV and reduced Ca2+ affinity at EF-IV, with moderate metal-dependent differences in affinity for the alphaIIb domain.
E172Q and E172D mutants showed no and weak Mg2+ binding at EF-IV, respectively, and each mutant had reduced Ca2+ affinity at EF-IV and showed moderate metal-dependent differences in affinity for the alphaIIb domain.
The CIB1 E172Q mutant shows no Mg2+ binding at EF-IV and reduced Ca2+ affinity at EF-IV, with moderate metal-dependent differences in affinity for the alphaIIb domain.
E172Q and E172D mutants showed no and weak Mg2+ binding at EF-IV, respectively, and each mutant had reduced Ca2+ affinity at EF-IV and showed moderate metal-dependent differences in affinity for the alphaIIb domain.
D127E and D127Q mutations disrupt alphaIIb binding by expanding the EF-III metal-binding loop and changing alpha-helix positions in EF-III.
These data, combined with a NMR-based structural comparison of the Mg2+- and Ca2+-loaded CIB1-alphaIIb peptide complexes, suggest that the D127E and D127Q mutations have a disruptive effect on alphaIIb binding since they expand the metal-binding loop and change the alpha-helix positions in EF-III.
Approval Evidence
1 source5 linked approval claimsfirst-pass slug ef-iii-metal-binding-site-of-cib1
EF-III is a mixed Mg2+/Ca2+-binding site
Source:
binding site propertysupports
CIB1 EF-III is a mixed Mg2+/Ca2+-binding site.
EF-III is a mixed Mg2+/Ca2+-binding site
Source:
evolutionary inferencesupports
Replacement of the ancestral Glu with Asp at the -Z position of EF-III increased CIB1 affinity for alphaIIb and shifted Ca2+ affinity into a range compatible with intracellular Ca2+ sensing.
upon replacement of the ancestral Glu with Asp at the -Z position of EF-III, CIB1 gained affinity for alphaIIb, and the Ca2+ affinity of CIB1 shifted into a range where the protein is able to act as an intracellular Ca2+ sensor
Source:
mutation effectsupports
The CIB1 D127E mutant has increased Mg2+ and Ca2+ affinity at EF-III, but alphaIIb-domain binding is too weak to observe.
A D127E mutant had increased Mg2+ and Ca2+ affinity at EF-III, but unexpectedly, the affinity for the alphaIIb domain was too low for binding to be observed.
Source:
mutation effectsupports
The CIB1 D127N mutant has reduced Mg2+ and Ca2+ affinity at EF-III but retains affinity for the alphaIIb domain.
A D127N mutant had reduced Mg2+ and Ca2+ affinity at EF-III but retained affinity for the alphaIIb domain.
Source:
structural mechanismsupports
D127E and D127Q mutations disrupt alphaIIb binding by expanding the EF-III metal-binding loop and changing alpha-helix positions in EF-III.
These data, combined with a NMR-based structural comparison of the Mg2+- and Ca2+-loaded CIB1-alphaIIb peptide complexes, suggest that the D127E and D127Q mutations have a disruptive effect on alphaIIb binding since they expand the metal-binding loop and change the alpha-helix positions in EF-III.
Source:
Comparisons
Source-backed strengths
The site is experimentally supported as a mixed Mg2+/Ca2+-binding EF-hand, and targeted substitutions at the -Z position produced interpretable shifts in both metal affinity and alphaIIb binding behavior. In particular, replacement of the ancestral Glu with Asp at EF-III increased alphaIIb affinity and shifted Ca2+ affinity into a range compatible with intracellular Ca2+ sensing, indicating functional tunability.
Ranked Citations
- 1.