Yuji Jinbo

Nef of HIV‐1 interacts directly with calcium‐bound calmodulin

It was recently found that the myristoyl group of CAP‐23/NAP‐22, a neuron‐specific protein kinase C substrate, is essential for the interaction between the protein and Ca2+‐bound calmodulin (Ca2+/CaM). Based on the N‐terminal amino acid sequence alignment of CAP‐23/NAP‐22 and other myristoylated proteins, including the Nef protein from human immunodeficiency virus (HIV), we proposed a new hypothesis that the protein myristoylation plays important roles in protein–calmodulin interactions. To investigate the possibility of direct interaction between Nef and calmodulin, we performed structural studies of Ca2+/CaM in the presence of a myristoylated peptide corresponding to the N‐terminal region of Nef. The dissociation constant between Ca2+/CaM and the myristoylated Nef peptide was determined to be 13.7 nM by fluorescence spectroscopy analyses. The NMR experiments indicated that the chemical shifts of some residues on and around the hydrophobic clefts of Ca2+/CaM changed markedly in the Ca2+/CaM‐Nef peptide complex with the molar ratio of 1:2. Correspondingly, the radius of gyration determined by the small angle X‐ray scattering measurements is 2–3 Å smaller that of Ca2+/CaM alone. These results demonstrate clearly that Nef interacts directly with Ca2+/CaM.

Solution X-ray scattering reveals a novel structure of calmodulin complexed with a binding domain peptide from the HIV-1 matrix protein p17.

The solution structures of complexes between calcium-saturated calmodulin (Ca (2+)/CaM) and a CaM-binding domain of the HIV-1 matrix protein p17 have been determined by small-angle X-ray scattering with use of synchrotron radiation as an intense and stable X-ray source. We used three synthetic peptides of residues 11-28, 26-47, and 11-47 of p17 to demonstrate the diversity of CaM-binding conformation. Ca (2+)/CaM complexed with residues 11-28 of p17 adopts a dumbbell-like structure at a molar ratio of 1:2, suggesting that the two peptides bind each lobe of CaM, respectively. Ca (2+)/CaM complexed with residues 26-47 of p17 at a molar ratio of 1:1 adopts a globular structure similar to the NMR structure of Ca (2+)/CaM bound to M13, which adopted a compact globular structure. In contrast to these complexes, Ca (2+)/CaM binds directly with both CaM-binding sites of residues 11-47 of p17 at a molar ratio of 1:1, which induces a novel structure different from known structures previously reported between Ca (2+)/CaM and peptide. A tertiary structural model of the novel structure was constructed using the biopolymer module of Insight II 2000 on the basis of the scattering data. The two domains of CaM remain essentially unchanged upon complexation. The hinge motions, however, occur in a highly flexible linker of CaM, in which the electrostatic residues 74Arg, 78Asp, and 82Glu interact with N-terminal electrostatic residues of the peptide (residues 12Glu, 15Arg, and 18Lys). The acidic residues in the N-terminal domain of CaM interact with basic residues in a central part of the peptide, thereby enabling the central part to change the conformations, while an acidic residue in the C-terminal domain interacts with two basic residues in the two helical sites of the peptide. The overall structure of the complex adopts an extended structure with the radius of gyration of 20.5 A and the interdomain distance of 34.2 A. Thus, the complex is principally stabilized by electrostatic interactions. The hydrophobic patches of Ca (2+)/CaM are not responsible for the binding with the hydrophobic residues in the peptide, suggesting that CaM plays a role to sequester the myristic acid moiety of p17.

Increase in the molecular weight and radius of gyration of apocalmodulin induced by binding of target peptide: evidence for complex formation

Small‐angle X‐ray scattering was used to investigate a complex state of apocalmodulin induced by the binding of a Ca2+/calmodulin‐dependent protein kinase IV calmodulin target site. Upon binding of the peptide, the molecular weight for apocalmodulin increased by 8.4%, which provides direct evidence for the formation of a calmodulin/target peptide complex. Comparison of the radius of gyration and Kratky plots of the apocalmodulin/peptide complex with those of apocalmodulin indicates that the overall conformation remains unchanged but the flexibility of the central linker decreases. An analysis of residue pairs between calmodulin and the target peptides suggests that the complex formation is induced by electrostatic interactions and subsequent van der Waals interactions.

Novel Mechanism of Regulation of Protein 4.1G Binding Properties Through Ca2+/Calmodulin-Mediated Structural Changes

Protein 4.1G (4.1G) is a widely expressed member of the protein 4.1 family of membrane skeletal proteins. We have previously reported that Ca2+-saturated calmodulin (Ca2+/CaM) modulates 4.1G interactions with transmembrane and membrane-associated proteins through binding to Four.one-ezrin–radixin–moesin (4.1G FERM) domain and N-terminal headpiece region (GHP). Here we identify a novel mechanism of Ca2+/CaM-mediated regulation of 4.1G interactions using a combination of small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy analyses. We document that GHP intrinsically disordered coiled structure switches to a stable compact structure upon binding of Ca2+/CaM. This dramatic conformational change of GHP inhibits in turn 4.1G FERM domain interactions due to steric hindrance. Based upon sequence homologies with the Ca2+/CaM-binding motif in protein 4.1R headpiece region, we establish that the 4.1G S71RGISRFIPPWLKKQKS peptide (pepG) mediates Ca2+/CaM binding. As observed for GHP, the random coiled structure of pepG changes to a relaxed globular shape upon complex formation with Ca2+/CaM. The resilient coiled structure of pepG, maintained even in the presence of trifluoroethanol, singles it out from any previously published CaM-binding peptide. Taken together, these results show that Ca2+/CaM binding to GHP, and more specifically to pepG, has profound effects on other functional domains of 4.1G.

Unfolding intermediate of a multidomain protein, calmodulin, in urea as revealed by small-angle X-ray scattering.

The denaturation of calmodulin (CaM) induced by urea has been studied by small‐angle X‐ray scattering, which is a direct way to evaluate the shape changes in a protein molecule. In the absence of Ca2+, the radii of gyration (R g) of CaM are 20.8±0.3 Å in the native state and about 34±1.0 Å in the unfolded state. The transition curve derived from Kratky plots indicates a bimodal transition via a stable unfolding intermediate around 2.5 M urea. In the presence of Ca2+ and in the presence of both Ca2+ and a target peptide, the R g values are 21.5±0.3 and 18.1±0.3 Å in the native state and 26.7±0.4 and 24.9±0.4 Å at 9 M urea, respectively. The results indicate that a stable unfolding intermediate still persists in 9 M urea. The present results suggest that the shape of unfolding intermediates is an asymmetric dumbbell‐like structure, one in the folded and one in the unfolded state.

Binding of trifluoperazine to apocalmodulin revealed by a combination of small-angle X-ray scattering and nuclear magnetic resonance

Small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) studies were performed to investigate the binding of trifluoperazine (TFP) to Ca2+-free calmodulin (apoCaM) with N- and C-terminal globular domains connected by a linker. The SAXS and NMR measurements were taken throughout the titration of TFP. The SAXS analyses indicate that the binding of TFP induces structural changes from a dumbbell shape to a compact globular shape in solution. The formation of the complete globular structure requires 5.0 added equivalents of TFP. An analysis of NMR chemical-shift changes indicates that the C-terminal domain of apoCaM is involved in the binding of TFP. The SAXS and NMR data reflect the high structural flexibility of apoCaM.

Relation between Huggins Constant and Compatibility of Binary Polymer Mixtures in the Aqueous Ternary Systems

We have classified a number of aqueous ternary systems containing two different polymers into three types by focusing on the deviation of the Huggins constantk′ from the additivity line. Systems of type I have negative deviations ofk′; the repulsive interaction between the two different polymers dominates. In systems of type II,k′ almost follows the additivity relation; the repulsive and attractive interactions between the two different polymers are balancing. Type III systems have positive deviations ofk′; the attractive interactions are relatively dominant. This classification of systems is supported by the fact that the positive and negative deviations ofk′ from the additivity line also correspond to the sign of interaction parameter between polymer 2 and 3, Δb23. Furthermore, we have verified the relatively high compatibility between dextran and poly(vinyl alcohol) by determining the binodal concentration of a liquid-liquid phase separation for a water/dextran/poly(vinyl alcohol) system, which is classified as type III. Thus, we found that the compositional dependence ofk′ closely relates to the compatibility of binary polymer mixtures in aqueous ternary systems.

Orientational correlation of liquid–crystalline polymer chains in isotropic solutions. I. Anisotropic light scattering

The orientational fluctuation in isotropic toluene and dichloromethane solutions of a stiff-chain polymer, poly(n-hexyl isocyanate) (PHIC), has been studied by anisotropic light scattering up to the vicinity of the isotropic–nematic phase separation region. The depolarized component ΔRθ,Hv of the Rayleigh ratio divided by the polymer concentration increased with increasing the polymer concentration. The ΔRθ,Hv data for different molecular weights were fitted almost quantitatively to the scaled particle theory for wormlike hard-spherocylinders with the hard-core diameter d, developed by incorporating the higher virial terms in the free energy. However, the fitted d value was appreciably smaller than that chosen to explain the experimental osmotic compressibilities and/or isotropic–nematic phase boundaries of the same systems. Then it was found that the “spinodal concentration” c*, where the isotropic phase becomes thermodynamically unstable, lies within the nematic region, in contrast with the prediction o...

Solution structure of Ca2+/calmodulin complexed with a lentivirus lytic peptide 1 reveals a novel mode of molecular recognition

Small-angle X-ray scattering was used to analyze the interaction of Ca2+/calmodulin (CaM) with a lentivirus lytic peptide 1 (LLP1) derived from the cytoplasmic tail of HIV-1 transmembrane glycoprotein. The synthetic peptide homologues of LLP1 were selected from three species of the glycoprotein: ENV_HV1A2, ENV_HV1B1 and ENV_HV1H2. Ca2+/CaM binds LLP1 with the truncation of three or ten residues and adopts almost the same globular structure as that of the complex with a peptide from myosin light chain kinase (MLCK), indicating that the Ca2+/CaM-binding site locates on the shorter sequence. Moreover, Ca2+/CaM binds a peptide with the opposite sequence and adopts almost the same globular structure as that in the original sequence. Taken together, the results provide evidence that LLP1 can bind to the N- and C-terminal lobes of CaM with a polarity opposite to that observed for the CaM–MLCK complex and the binding mode of Ca2+/CaM molecular recognition is well preserved despite the sequence variation in the three species, suggesting that this region of the transmembrane glycoprotein is important to viral replication.