The small bilobal calcium regulatory protein calmodulin (CaM) activates numerous target enzymes in response to transient changes in intracellular calcium concentrations. Binding of calcium to the two helix-loop-helix calcium-binding motifs in each of the globular domains induces conformational changes that expose a methionine-rich hydrophobic patch on the surface of each domain of the protein, which it uses to bind to peptide sequences in its target enzymes. Although these CaM-binding domains typically have little sequence identity, the positions of several bulky hydrophobic residues are often conserved, allowing for classification of CaM-binding domains into recognition motifs, such as the 1-14 and 1-10 motifs. For calcium-independent binding of CaM, a third motif known as the IQ motif is also common. Many CaM-peptide complexes have globular conformations, where CaM's central linker connecting the two domains unwinds, allowing the protein to wrap around a single predominantly alpha-helical target peptide sequence. However, novel structures have recently been reported where the conformation of CaM is highly dissimilar to these globular complexes, in some instances with less than a full compliment of bound calcium ions, as well as novel stoichiometries. Furthermore, many divergent CaM isoforms from yeast and plant species have been discovered with unique calcium-binding and enzymatic activation characteristics compared to the single CaM isoform found in mammals.

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Summary While engagement of the inhibitory Fcγ-Receptor (FcγR) IIB is an absolute requirement for in vivo antitumor activity of agonistic mouse anti-CD40 monoclonal antibodies (mAbs), a similar requirement for human mAbs has been disputed. By using a mouse model humanized for its FcγRs and CD40, we revealed that FcγRIIB-engagement is essential for the activity of the human CD40 mAbs, while engagement of the activating FcγRIIA inhibits this activity. By engineering Fc variants with selective enhanced binding to FcγRIIB, but not to FcγRIIA, significantly improved antitumor immunity was observed. These findings highlight the necessity of optimizing the Fc domain for this class of therapeutic antibodies by using appropriate pre-clinical models that accurately reflect the unique affinities and cellular expression of human FcγR.

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.

Calcium-and integrin-binding protein 1 (CIB1) regulates platelet aggregation in hemostasis through a specific interaction with the ␣IIb cytoplasmic domain of platelet integrin ␣IIb␤ 3 . 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 ␣IIb cytoplasmic domain. NMR spectroscopy experiments using perdeuterated CIB1 together with heteronuclear nuclear Overhauser effect experiments have revealed a well folded ␣-helical structure for both the ligand-free and ␣IIb-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 ␣IIb-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 ␣IIb cytoplasmic domain, suggesting a novel mechanism to increase binding specificity.The platelet-specific heterodimeric transmembrane integrin receptor ␣IIb␤ 3 plays a central role in hemostasis and thrombosis (1). At the site of vascular injury, platelet agonists such as thrombin trigger "inside-out" signaling events that activate ␣IIb␤ 3 , resulting in ligand binding by the integrin extracellular domains, integrin cross-linking, and ultimately, platelet aggregation. Ligand occupancy also generates "outside-in" signals that lead to granular secretion of ADP, cytoskeletal reorganization, and platelet spreading (2). The small EF-hand calciumbinding protein CIB1, 4 (calcium-and integrin-binding protein 1, also known as CIB, calmyrin, KIP) binds specifically to the ␣IIb cytoplasmic domain (3), and the interaction has been implicated in both inside-out and outside-in signaling events (4 -6). The binding of CIB1 to synthetic ␣IIb peptides can occur in vitro with a dissociation constant in the high nanomolar range (7,8); however, the mechanistic details of the interaction are not well understood. Because inappropriate platelet activation is a major contributor to cardiovascular disease (9), understanding the interaction between CIB1 and ␣IIb could be an important step toward the development of novel anti-platelet therapeutics.CIB1 shares significant sequence homology with calcineurin B (CnB), calcineurin homologous protein-1, and the neuronal calcium sensor (NCS) family of EF-hand proteins. Like these proteins, CIB1 is myristoylated on its N-terminal glycine residue and is membrane-associated in vivo (10, 11). However, myristoylation is not required for ␣IIb binding (7,8,10). Recent x-ray crystal structures (Protein Data Bank codes 1XO5 and 1Y1A) have shown that like its homologs, calcium-bound CIB1 (Ca 2ϩ -CIB1) folds into Nand C-terminal globular domains, each composed of two EF-hand motifs, with extended N-and C-terminal regions (12, 13). Ca 2ϩ is bound to th...