scholarly journals Homology modelling of integrin EF-hands. Evidence for widespread use of a conserved cation-binding site

1992 ◽  
Vol 285 (1) ◽  
pp. 325-331 ◽  
Author(s):  
D S Tuckwell ◽  
A Brass ◽  
M J Humphries
Keyword(s):  
Ligand Binding ◽  
Homology Modelling ◽  
Cation Binding ◽  
Binding Motif ◽  
Binding Model ◽  
Bivalent Cation ◽  
Cation Binding Site ◽  
Ef Hand ◽  
Ef Hands

Integrin alpha-subunits contain three or four peptide sequences that are similar to the EF-hand, a 13-residue bivalent cation-binding motif found in calmodulin and parvalbumin. The integrin sequences differ from classical EF-hands in that they lack a co-ordinating residue at position 12. One hypothesis to explain integrin-ligand binding is that aspartate-containing recognition sequences in integrin ligands, which bind at or near to the EF-hand-like sequences, may take the place of the missing residue and co-ordinate directly to the bound cation. In this report, homology modelling of integrin EF-hand-like sequences has been performed using the X-ray structure of calmodulin as a template in order to assess the functional activity of the integrin sequences. In the calmodulin-integrin hybrid structures, integrin EF-hand-like sequences were able to retain cations whereas control sequences did not. Structural analyses demonstrated that the integrin sequences in the hybrid proteins closely resembled conventional EF-hands. The integrin sequences are therefore highly likely to bind Ca2+ ions in vivo, a prerequisite for the ligand-binding model. Database searching with a matrix derived from known integrin EF-hand-like sequences has been used to identify other proteins containing the integrin EF-hand-like motif. Annexin V (anchorin CII), atrial natriuretic peptide receptors and the 70 kDa heat-shock protein were identified by the matrix; the functions of these proteins are known from previous studies to be bivalent cation-dependent. These findings suggest that the integrin EF-hand-like sequence may be a more common motif than originally thought.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

2020 ◽  
Vol 477 (1) ◽  
pp. 173-189 ◽  
Author(s):  
Marco Pedretti ◽  
Carolina Conter ◽  
Paola Dominici ◽  
Alessandra Astegno
Keyword(s):  
Excision Repair ◽  
Complex Structure ◽  
Binding Motif ◽  
C Terminus ◽  
Repair Protein ◽  
Nucleotide Excision ◽  
Ef Hand ◽  
Molecular Determinants ◽  
Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

Structures and metal-ion-binding properties of the Ca2+-binding helix–loop–helix EF-hand motifs

2007 ◽  
Vol 405 (2) ◽  
pp. 199-221 ◽  
Author(s):  
Jessica L. Gifford ◽  
Michael P. Walsh ◽  
Hans J. Vogel
Keyword(s):  
Metal Ion ◽  
Molten Globule ◽  
Family Members ◽  
Binding Motif ◽  
Metal Ion Binding ◽  
Protein Target ◽  
Helix Loop Helix ◽  
Wide Range ◽  
Ef Hand ◽  
Ef Hands

The ‘EF-hand’ Ca2+-binding motif plays an essential role in eukaryotic cellular signalling, and the proteins containing this motif constitute a large and functionally diverse family. The EF-hand is defined by its helix–loop–helix secondary structure as well as the ligands presented by the loop to bind the Ca2+ ion. The identity of these ligands is semi-conserved in the most common (the ‘canonical’) EF-hand; however, several non-canonical EF-hands exist that bind Ca2+ by a different co-ordination mechanism. EF-hands tend to occur in pairs, which form a discrete domain so that most family members have two, four or six EF-hands. This pairing also enables communication, and many EF-hands display positive co-operativity, thereby minimizing the Ca2+ signal required to reach protein saturation. The conformational effects of Ca2+ binding are varied, function-dependent and, in some cases, minimal, but can lead to the creation of a protein target interaction site or structure formation from a molten-globule apo state. EF-hand proteins exhibit various sensitivities to Ca2+, reflecting the intrinsic binding ability of the EF-hand as well as the degree of co-operativity in Ca2+ binding to paired EF-hands. Two additional factors can influence the ability of an EF-hand to bind Ca2+: selectivity over Mg2+ (a cation with very similar chemical properties to Ca2+ and with a cytoplasmic concentration several orders of magnitude higher) and interaction with a protein target. A structural approach is used in this review to examine the diversity of family members, and a biophysical perspective provides insight into the ability of the EF-hand motif to bind Ca2+ with a wide range of affinities.

scholarly journals Amyloid Assembly Endows Gad m 1 with Biomineralization Properties

2018 ◽  
Author(s):  
Milagros Castellanos ◽  
Rosa Rodríguez-Pérez ◽  
María Gasset
Keyword(s):  
Calcium Carbonate ◽  
Atlantic Cod ◽  
Fish Muscle ◽  
Binding Motif ◽  
Beta Strand ◽  
Ef Hand ◽  
Acidic Conditions ◽  
Ef Hands ◽  
Amyloid Assembly

Acid proteins capable of nucleating Ca2+ and displaying aggregation capacity play key roles in the formation of calcium carbonate biominerals. EF-hands are among the largest Ca2+-binding motif in proteins. Gad m 1, an Atlantic cod β-parvalbumin isoform, is a monomeric EF-hand protein that acts as a Ca2+ buffer in fish muscle and is able to form amyloids under acidic conditions. Since nucleating Ca2+ protein have a propensity to form extended β-strand structures, we wondered whether amyloid assemblies of a protein containing refolded EF-hand motifs were able to influence the in vitro calcium carbonate crystallization. Here we have used the Gad m 1 chain as model to generate monomeric and amyloid assemblies and analyze their effect on in vitro calcite formation. We found that only amyloid assemblies alter calcite morphology.

Binding of oxovanadium ions to the major and minor grooves of DNA duplex: stability and structural models

2006 ◽  
Vol 84 (5) ◽  
pp. 677-683 ◽  
Author(s):  
A. Ahmed Ouameur ◽  
H. Arakawa ◽  
H.A. Tajmir-Riahi
Keyword(s):  
Human Fibroblasts ◽  
Binding Constants ◽  
Chemical Carcinogenesis ◽  
Cation Binding ◽  
Molar Ratios ◽  
Cation Binding Site ◽  
Thymine Adenine ◽  
Group A ◽  
Dna Strand

Vanadate induces DNA strand breaks in cultured human fibroblasts at doses that are relative to the occupational exposure. Oxovanadium compounds also exert preventive effects against chemical carcinogenesis in animals and form complexes with DNA in vivo. This study was designed to examine the interaction of calf-thymus DNA with VO2+ and VO3¯ ions in aqueous solution at physiological pH, with a constant DNA concentration of 12.5 mmol/L and vanadium–DNA (phosphate) molar ratios (r) of 1:160 to 1:2. Capillary electrophoresis and Fourier transform infrared difference spectroscopy were used to determine the cation binding site, the binding constant, the helix stability, and DNA conformation in the oxovanadium–DNA complexes. Structural analysis showed that VO2+ binds DNA through guanine and adenine N-7 atoms and the backbone PO2 group with apparent binding constants of KG = 8.8 × 105 (mol/L)–1 and KA = 3.4 × 105 (mol/L)–1. The VO3¯ shows weaker binding through thymine, adenine, and guanine bases, with K = 1.9 × 104 (mol/L)–1 and no interaction with the backbone phosphate group. A partial B-to-A DNA transition occurred upon VO–DNA complexation, while DNA remains in the B-family structure in the VO3¯ complexes.

scholarly journals The Ca(2+)-binding domains in non-muscle type alpha-actinin: biochemical and genetic analysis.

1993 ◽  
Vol 121 (3) ◽  
pp. 599-606 ◽  
Author(s):  
W Witke ◽  
A Hofmann ◽  
B Köppel ◽  
M Schleicher ◽  
A A Noegel
Keyword(s):  
Biochemical Data ◽  
Actin Binding ◽  
Regulatory Function ◽  
Cross Linking ◽  
Abnormal Phenotype ◽  
Ef Hand ◽  
Ef Hands ◽  
Overlay Assay

Dictyostelium alpha-actinin is a Ca(2+)-regulated F-actin cross-linking protein. To test the inhibitory function of the two EF hands, point mutations were introduced into either one or both Ca(2+)-binding sites. After mutations, the two EF hands were distinguishable with respect to their regulatory activities. Inactivation of EF hand I abolished completely the F-actin cross-linking activity of Dictyostelium discoideum alpha-actinin but Ca2+ binding by EF hand II was still observed in a 45Ca2+ overlay assay. In contrast, after mutation of EF hand II the molecule was still active and inhibited by Ca2+; however, approximately 500-fold more Ca2+ was necessary for inhibition and 45Ca2+ binding could not be detected in the overlay assay. These data indicate that EF hand I has a low affinity for Ca2+ and EF hand II a high affinity, implying a regulatory function of EF hand I in the inhibition of F-actin cross-linking activity. Biochemical data is presented which allows us to distinguish two functions of the EF hand domains in D. discoideum alpha-actinin: (a) at the level of the EF-hands, the Ca(2+)-binding affinity of EF hand I was increased by EF hand II in a cooperative manner, and (b) at the level of the two subunits, the EF hands acted as an on/off switch for actin-binding in the neighboring subunit. To corroborate in vitro observations in an in vivo system we tried to rescue the abnormal phenotype of a mutant (Witke, W., M. Schleicher, A. A. Noegel. 1992. Cell. 68:53-62) by introducing the mutated alpha-actinin cDNAs. In agreement with the biochemical data, only the molecule modified in EF hand II could rescue the abnormal phenotype. Considering the fact that the active construct is "always on" because it requires nonphysiological, high Ca2+ concentrations for inactivation, it is interesting to note that an unregulated alpha-actinin was able to rescue the mutant phenotype.

scholarly journals Structures of SALSA/DMBT1 SRCR domains reveal the conserved ligand-binding mechanism of the ancient SRCR fold

2020 ◽  
Vol 3 (4) ◽  
pp. e201900502 ◽  
Author(s):  
Martin P Reichhardt ◽  
Vuokko Loimaranta ◽  
Susan M Lea ◽  
Steven Johnson
Keyword(s):  
Ligand Binding ◽  
Scavenger Receptor ◽  
Structural Data ◽  
Cation Binding ◽  
Binding Mechanism ◽  
Ligand Interaction ◽  
Srcr Domain ◽  
Binding Interface ◽  
Cation Binding Site ◽  
High Level

The scavenger receptor cysteine-rich (SRCR) family of proteins comprises more than 20 membrane-associated and secreted molecules. Characterised by the presence of one or more copies of the ∼110 amino-acid SRCR domain, this class of proteins have widespread functions as antimicrobial molecules, scavenger receptors, and signalling receptors. Despite the high level of structural conservation of SRCR domains, no unifying mechanism for ligand interaction has been described. The SRCR protein SALSA, also known as DMBT1/gp340, is a key player in mucosal immunology. Based on detailed structural data of SALSA SRCR domains 1 and 8, we here reveal a novel universal ligand-binding mechanism for SALSA ligands. The binding interface incorporates a dual cation-binding site, which is highly conserved across the SRCR superfamily. Along with the well-described cation dependency on most SRCR domain–ligand interactions, our data suggest that the binding mechanism described for the SALSA SRCR domains is applicable to all SRCR domains. We thus propose to have identified in SALSA a conserved functional mechanism for the SRCR class of proteins.

scholarly journals The structure of SALSA/DMBT1 SRCR domains reveal the conserved ligand-binding mechanism of the ancient SRCR-fold

2019 ◽  
Author(s):  
Martin P. Reichhardt ◽  
Vuokko Loimaranta ◽  
Susan M. Lea ◽  
Steven Johnson
Keyword(s):  
Ligand Binding ◽  
Scavenger Receptor ◽  
Cation Binding ◽  
Binding Mechanism ◽  
Ligand Interaction ◽  
Srcr Domain ◽  
Binding Interface ◽  
Cation Binding Site ◽  
Ligand Interactions ◽  
High Level

AbstractThe scavenger receptor cysteine-rich (SRCR) family of proteins comprise more than 20 membrane-associated and secreted molecules. Characterised by the presence of one or more copies of the ~110 amino acid SRCR domain, this class of proteins have widespread functions as anti-microbial molecules, scavenger- and signalling-receptors. Despite the high level of structural conservation of SRCR domains, no molecular basis for ligand interaction has been described. The SRCR protein SALSA, also known as dmbt1/gp340, is a key player in mucosal immunology. Based on detailed structures of the SALSA SRCR domains 1 and 8, we here reveal a novel universal ligand binding mechanism for SALSA ligands. The binding interface incorporates a dual cation binding site, which is highly conserved across the SRCR super family. Along with the well-described cation dependency on most SRCR domain-ligand interactions, our data suggest that the binding mechanism described for the SALSA SRCR domains is applicable to all SRCR domains. We thus propose to have identified in SALSA a conserved functional mechanism for ligand recognition by the SRCR class of proteins.

scholarly journals Differences between cotranscriptional and free riboswitch folding

2013 ◽  
Vol 42 (4) ◽  
pp. 2687-2696 ◽  
Author(s):  
Benjamin Lutz ◽  
Michael Faber ◽  
Abhinav Verma ◽  
Stefan Klumpp ◽  
Alexander Schug
Keyword(s):  
Ligand Binding ◽  
Messenger Rna ◽  
Kinetic Monte Carlo ◽  
Coarse Grained ◽  
Transcription Rate ◽  
Binding Model ◽  
Rna Sensors ◽  
Transcript Elongation ◽  
Dynamics Simulations

Abstract Riboswitches are part of noncoding regions of messenger RNA (mRNA) that act as RNA sensors regulating gene expression of the downstream gene. Typically, one out of two distinct conformations is formed depending on ligand binding when the transcript leaves RNA polymerase (RNAP). Elongation of the RNA chain by RNAP, folding and binding all occurs simultaneously and interdependently on the seconds’ timescale. To investigate the effect of transcript elongation velocity on folding for the S-adenosylmethionine (SAM)-I and adenine riboswitches we employ two complementary coarse-grained in silico techniques. Native structure-based molecular dynamics simulations provide a 3D, atomically resolved model of folding with homogenous energetics. Energetically more detailed kinetic Monte Carlo simulations give access to longer timescale by describing folding on the secondary structure level and feature the incorporation of competing aptamer conformations and a ligand-binding model. Depending on the extrusion scenarios, we observe and quantify different pathways in structure formation with robust agreements between the two techniques. In these scenarios, free-folding riboswitches exhibit different folding characteristics compared with transcription-rate limited folding. The critical transcription rate distinguishing these cases is higher than physiologically relevant rates. This result suggests that in vivo folding of the analyzed SAM-I and adenine riboswitches is transcription-rate limited.

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