Domain stability and metal-induced folding of calcium- and integrin-binding protein 1

It is widely accepted that a pair of EF-hands is the functional unit of typical four EF-hand proteins such as calmodulin or troponin C. In this work we investigate the structure and stability of the four EF-hand domains in the related protein calcium- and integrin-binding protein 1 (CIB1) in the presence and absence of Mg2+ or Ca2+, to determine if similar EF-hand interactions occur. The backbone structure and flexibility of CIB1 were first studied by NMR spectroscopy, and these studies were complimented with steady-state fluorescence spectroscopy and chemical denaturation experiments using mutant CIB1 proteins having single Trp reporter groups in each of the four EF-hand domains EF-I (F34W), EF-II (F91W), EF-III (L128W), and EF-IV (F173W). We find that Mg2+-CIB1 adopts a well-folded structure similar to Ca2+-CIB1, except for some conformational heterogeneity in the C-terminal EF-IV domain. The structure of apo-CIB1 is significantly more dynamic, especially within EF-II, EF-III, and a partially unfolded EF-IV region, but the N-terminal EF-I region of apo-CIB1 has a well-ordered and more stable structure. The data reveal significant communication between the N- and C-lobes of CIB1, and show that transient intermediate conformations are formed along the unfolding pathway for each form of the protein. Collectively the data demonstrate that the communication between the paired EF-hand domains as well as between the N- and C-lobes of CIB1 is distinct from the ancestral proteins calmodulin and troponin C, which might be important for the unique function of CIB1 in numerous biological processes.     

Metal ion binding properties and conformational states of calcium- and integrin-binding protein

Calcium- and integrin-binding protein (CIB) is a novel member of the helix-loop-helix family of regulatory calcium-binding proteins which likely has a specific function in hemostasis through its interaction with platelet integrin alphaIIbbeta(3). The significant amino acid sequence homology between CIB and other regulatory calcium-binding proteins such as calmodulin, calcineurin B, and recoverin suggests that CIB may undergo a calcium-induced conformational change; however, the mechanism of calcium binding and the details of a structural change have not yet been investigated. Consequently, we have performed a variety of spectroscopic and microcalorimetric studies of CIB to determine its calcium binding characteristics, and the subsequent conformational changes that occur. Furthermore, we provide the first evidence for magnesium binding to CIB and determine the structural consequences of this interaction. Our results indicate that in the absence of any bound metal ions, apo-CIB adopts a folded yet highly flexible molten globule-like structure. Both calcium and magnesium binding induce conformational changes which stabilize both the secondary and tertiary structure of CIB, resulting in considerable increases in the thermal stability of the proteins. CIB was found to bind two Ca(2+) ions in a sequential manner with dissociation constants (K(d)) near 0.54 and 1.9 microM for sites EF-4 and EF-3, respectively. In contrast, CIB bound only one Mg(2+) ion to EF-3 with a K(d) near 120 microM. Together, our results suggest that CIB may exist in multiple structural and metal ion-bound states in vivo which may play a role in its regulation of target proteins such as platelet integrin.     

Assessment of structural and functional similarities and differences between caeca of the bluegill

Bluegill Lepomis macrochirus possess six to eight gastrointestinal caeca, >70% having seven. The terminal caeca toward abdominal edge (C6, C7) were always larger than the terminal caeca toward the vertebral column (C1, C2) and middle caeca (C3, C4, C5). The caeca varied in relative thickness of the histological layers and lumen space. The terminal caeca (C1, C2, C6, C7) have anatomical positional advantage over, and possess more contents, than the middle ones (C3, C4, C5) in both field and laboratory conditions. Hence, all caeca in bluegill may have the same function but their functional potentials are not the same. Two factors, size of caeca and anatomical position of caeca, apparently work synergistically to cause variation in their functional efficiency.     

Calcium- and magnesium-dependent interactions between calcium- and integrin-binding protein and the integrin alphaIIb cytoplasmic domain

Calcium- and integrin-binding protein (CIB) is a small EF-hand calcium-binding protein that is involved in hemostasis through its interaction with the alphaIIb cytoplasmic domain of integrinalphaIIbbeta(3). We have previously demonstrated that CIB lacks structural stability in the absence of divalent metal ions but that it acquires a well-folded conformation upon addition of Ca(2+) or Mg(2+). Here, we have used fluorescence spectroscopy, NMR spectroscopy, and isothermal titration calorimetry to demonstrate that both Ca(2+)-bound CIB (Ca(2+)-CIB) and the Mg(2+)-bound protein (Mg(2+)-CIB) bind with high affinity and through a similar mechanism to alphaIIb cytoplasmic domain peptides, but that metal-free CIB (apo-CIB) binds in a different manner. The interactions are thermodynamically distinct for Ca(2+)-CIB and Mg(2+)-CIB, but involve hydrophobic interactions in each case. Since the Mg(2+) concentration inside the cell is sufficient to saturate CIB at all times, our results imply that CIB would be capable of binding to the alphaIIb cytoplasmic domain independent of an intracellular Ca(2+) stimulus in vivo. This raises the question of whether CIB can act as a Ca(2+) sensor in alphaIIbbeta(3) signaling or if other regulatory mechanisms such as fibrinogen-induced conformational changes in alphaIIbbeta(3), post-translational modifications, or the binding of other accessory proteins mediate the interactions between CIB and alphaIIbbeta(3). Differences in NMR spectra do suggest, however, that Ca(2+)-binding to the Mg(2+)- CIB-alphaIIb complex induces subtle structural changes that could further modulate the activity of alphaIIbbeta(3).     

The crystal structure of calcium- and integrin-binding protein 1: insights into redox regulated functions

Calcium- and integrin-binding protein 1 (CIB1) is involved in the process of platelet aggregation by binding the cytoplasmic tail of the alpha(IIb) subunit of the platelet-specific integrin alpha(Iib)beta(3). Although poorly understood, it is widely believed that CIB1 acts as a global signaling regulator because it is expressed in many tissues that do not express integrin alpha(Iib)beta(3). We report the structure of human CIB1 to a resolution of 2.3 A, crystallized as a dimer. The dimer interface includes an extensive hydrophobic patch in a crystal form with 80% solvent content. Although the dimer form of CIB1 may not be physiologically relevant, this intersub-unit surface is likely to be linked to alpha(IIb) binding and to the binding of other signaling partner proteins. The C-terminal domain of CIB1 is structurally similar to other EF-hand proteins such as calmodulin and calcineurin B. Despite structural homology to the C-terminal domain, the N-terminal domain of CIB1 lacks calcium-binding sites. The structure of CIB1 revealed a complex with a molecule of glutathione in the reduced state bond to the N-terminal domain of one of the two subunits poised to interact with the free thiol of C35. Glutathione bound in this fashion suggests CIB1 may be redox regulated. Next to the bound GSH, the orientation of residues C35, H31, and S48 is suggestive of a cysteine-type protein phosphatase active site. The potential enzymatic activity of CIB1 is discussed and suggests a mechanism by which it regulates a wide variety of proteins in cells in addition to platelets.     

Structures of the platelet calcium- and integrin-binding protein and the alphaIIb-integrin cytoplasmic domain suggest a mechanism for calcium-regulated recognition; homology modelling and NMR studies

Calcium- and integrin-binding protein (CIB) binds to the 20-residue alphaIIb cytoplasmic domain of platelet alphaIIbbeta3 integrin. Amino acid sequence similarities with calmodulin (CaM) and calcineurin B (CnB) allowed the construction of homology-based models of calcium-saturated CIB as well as apo-CIB. In addition, the solution structure of the alphaIIb cytoplasmic domain in 45% aqueous trifluoroethanol was solved by conventional two-dimensional NMR methods. The models indicate that the N-terminal domain of CIB possesses a number of positively charged residues in its binding site that could interact with the acidic carboxy-terminal LEEDDEEGE sequence of alphaIIb. The C-terminal domain of CIB seems well-suited to bind the sequence WKVGFFKR, which forms a well-structured alpha helix; this is analogous to calmodulin and calcineurin B, which also bind alpha helices. Similarities between the C-terminal domains of CIB and calmodulin suggest that binding of CIB to the cytoplasmic domain of alphaIIb may be affected by fluctuations in the intracellular calcium concentration.     

Membrane protein reconstitution for functional and structural studies

Membrane proteins are involved in various critical biological processes,and studying membrane proteins represents a major challenge in protein biochemistry.As shown by both structural and functional studies,the membrane environment plays an essential role for membrane proteins.In vitro studies are reliant on the successful reconstitution of membrane proteins.This review describes the interaction between detergents and lipids that aids the understanding of the reconstitution processes.Then the techniques of detergent removal and a few useful techniques to refine the formed proteoliposomes are reviewed.Finally the applications of reconstitution techniques to study membrane proteins involved in Ca2+ signaling are summarized.     

The interaction between calcium- and integrin-binding protein 1 and the alphaIIb integrin cytoplasmic domain involves a novel C-terminal displacement mechanism

Calcium- and integrin-binding protein 1 (CIB1) regulates platelet aggregation in hemostasis through a specific interaction with the alphaIIb cytoplasmic domain of platelet integrin alphaIIbbeta3. In this work we report the structural characteristics of CIB1 in solution and the mechanistic details of its interaction with a synthetic peptide derived from the alphaIIb cytoplasmic domain. NMR spectroscopy experiments using perdeuterated CIB1 together with heteronuclear nuclear Overhauser effect experiments have revealed a well folded alpha-helical structure for both the ligand-free and alphaIIb-bound forms of the protein. Residual dipolar coupling experiments have shown that the N and C domains of CIB1 are positioned side by side, and chemical shift perturbation mapping has identified the alphaIIb-binding site as a hydrophobic channel spanning the entire C domain and part of the N domain. Data obtained with a truncated version of CIB1 suggest that the extreme C-terminal end of the protein weakly interacts with this channel in the absence of a biological target, but it is displaced by the alphaIIb cytoplasmic domain, suggesting a novel mechanism to increase binding specificity.     

Intermittent control models of human standing: similarities and differences

Two architectures of intermittent control are compared and contrasted in the context of the single inverted pendulum model often used for describing standing in humans. The architectures are similar insofar as they use periods of open-loop control punctuated by switching events when crossing a switching surface to keep the system state trajectories close to trajectories leading to equilibrium. The architectures differ in two significant ways. Firstly, in one case, the open-loop control trajectory is generated by a system-matched hold, and in the other case, the open-loop control signal is zero. Secondly, prediction is used in one case but not the other. The former difference is examined in this paper. The zero control alternative leads to periodic oscillations associated with limit cycles; whereas the system-matched control alternative gives trajectories (including homoclinic orbits) which contain the equilibrium point and do not have oscillatory behaviour. Despite this difference in behaviour, it is further shown that behaviour can appear similar when either the system is perturbed by additive noise or the system-matched trajectory generation is perturbed. The purpose of the research is to come to a common approach for understanding the theoretical properties of the two alternatives with the twin aims of choosing which provides the best explanation of current experimental data (which may not, by itself, distinguish beween the two alternatives) and suggesting future experiments to distinguish beween the two alternatives.     

Contra-regulation of calcium- and integrin-binding protein 1-induced cell migration on fibronectin by PAK1 and MAP kinase signaling

Calcium- and integrin-binding protein 1 (CIB1) has been shown to be involved in cell spreading and migration. The signaling events regulated by CIB1 during cell migration are poorly understood. Here we found that accumulation of CIB1 at the tip of the filopodia requires an intact cytoskeleton. Depletion of CIB1 using shRNA affects formation of FAK- and phosphotyrosine-rich focal adhesions without affecting stress fiber formation. Overexpression of CIB1 results in cell migration on fibronectin and Erk1/2 MAP kinase activation. CIB1-induced cell migration is dependent upon Erk1/2 activation, since it is inhibited by the MEK-specific inhibitor PD98059. Furthermore, CIB1-induced cell migration, as well as Erk1/2 activation, is dependent on PKC, Src family kinases as well as PI-3 kinase as it is inhibited by bisindolylmaleimide 1, PP2, and wortmannin, respectively, in a dose-dependent manner. Co-expression of dominant-negative Cdc42 completely abolished CIB1-induced cell migration. Additionally, co-expression of constitutively active, but not dominant negative PAK1, a CIB1 binding protein, inhibited CIB1-induced cell migration. These results suggest that CIB1 positively regulates cell migration and is necessary for the recruitment of FAK to the focal adhesions. Furthermore, CIB1-induced cell migration is dependent on MAP kinase signaling and its function is attenuated by PAK1.     

The Bacillus subtilis regulator protein SpoIIE shares functional and structural similarities with eukaryotic protein phosphatases 2C

Dephosphorylation of SpoIIAA-P by SpoIIE is strictly dependent on the presence of the bivalent metal ions Mn2+ or Mg2+. Replacement by Ala of one of the four Asp residues, invariant in all representatives of protein phosphatase 2C, completely abolished the SpoIIE phosphatase activity in vitro, whilst replacement of the Asp residues by another acidic amino acid, Glu, had varying effects on the activities of the resulting mutated proteins. D610E and D795E exhibited some residual activity while D628E and D745E were without enzymatic activity. The results suggest that the functional model in which metal-associated water molecules are involved in the dephosphorylation reaction catalyzed by human protein phosphatase 2C alpha can also be applied to the bacterial protein phosphatase 2C-like protein.     

Centrosomal localization of cyclins E and A: Structural similarities and functional differences

Recent identification of the modular CLS motifs responsible for cyclins A and E localization on centrosomes has revealed a tight linkage between the nuclear and centrosomal cycles. These G₁/S cyclins must localize on the centrosome in order for DNA replication to occur in the nucleus, whereas essential DNA replication factors also function on the centrosome to prevent centrosome overduplication. Both events are dependent on the presence of an intact CLS within each cyclin. Here we compare the cyclins A and E CLSs at the structural and functional levels and identify a new cyclin A CLS mutant that disrupts all CLS functions and reduces the affinity of cyclin A for Cdk2. Analysis of interactions of the CLS motif within the cyclin molecules highlights the importance of the cyclin CBOX1 region for Cdk2 binding.Key words: cyclin A, cyclin E, Cdk2, centrosome, CLS, PSTAIRE, DNA synthesis     

RNA and protein 3D structure modeling: similarities and differences

In analogy to proteins, the function of RNA depends on its structure and dynamics, which are encoded in the linear sequence. While there are numerous methods for computational prediction of protein 3D structure from sequence, there have been very few such methods for RNA. This review discusses template-based and template-free approaches for macromolecular structure prediction, with special emphasis on comparison between the already tried-and-tested methods for protein structure modeling and the very recently developed “protein-like” modeling methods for RNA. We highlight analogies between many successful methods for modeling of these two types of biological macromolecules and argue that RNA 3D structure can be modeled using “protein-like” methodology. We also highlight the areas where the differences between RNA and proteins require the development of RNA-specific solutions.     

Avidin related protein 2 shows unique structural and functional features among the avidin protein family

Background  

The chicken avidin gene family consists of avidin and several avidin related genes (AVRs). Of these gene products, avidin is the best characterized and is known for its extremely high affinity for D-biotin, a property that is utilized in numerous modern life science applications. Recently, the AVR genes have been expressed as recombinant proteins, which have shown different biotin-binding properties as compared to avidin.     

Three human interferon-alpha 2 subvariants disclose structural and functional differences.

The human interferon-alpha 2 subvariants 2a, 2b and 2c differ by only one or two amino acids at positions 23 and/or 34 of the mature protein. In this study, the coding regions of the three interferon-alpha 2 subvariants were derived from the cDNA of interferon-alpha 2c by site-directed in vitro mutagenesis. The interferon-alpha subvariants were synthesized using the same Escherichia coli strain for production and were subsequently purified. Comparative studies revealed that they differ significantly in their biological and antigenic properties. Therefore, amino acid positions 23 and 34 seem to be crucial for structure/function of human interferon-alpha. Furthermore, the study points to the importance of defining, whether such minor structural variants of naturally occurring polypeptides represent functional variants.