IntroductionPlatelet glycoprotein (GP) Ib-IX-V complex binds to von Willebrand factor (VWF) deposited at the injured blood vessel wall, mediating initial platelet rolling and adhesion to the injury site. Ligation of VWF to the GP Ib␣ subunit in the complex also transmits a signal to the platelet that leads to platelet activation and aggregation. 1,2 In addition to mediating outside-in signals, the GP Ib-IX-V complex is also capable of transmitting intracellular signals to the outside. [3][4][5][6][7] How this receptor complex mediates signaling in both directions is not clear, but recent work has suggested the likely interaction between the Ib␣ and Ib␤ subunits as a potential key element of the mechanism. [8][9][10] Thus, understanding the interaction between GP Ib␣ and GP Ib␤, and in general the organizing principle of the GP Ib-IX-V complex, will help to unveil the mechanism underlying transmembrane signaling mediated by the complex.It is widely accepted that the GP Ib-IX-V complex contains 7 subunits composed of 4 different polypeptides: Ib␣, Ib␤, IX, and V with a respective stoichiometry of 2:2:2:1. [11][12][13][14][15] Each subunit is a type I transmembrane protein. A disulfide bond links GP Ib␣ and GP Ib␤, forming a complex known as GP Ib, that in turn interacts noncovalently with GP IX and GP V to generate the Ib-IX-V complex. The Ib␣ extracellular domain contains 9 Cys residues, 7 of which reside in the N-terminal domain that contains the binding site for VWF. The N-terminal domain contains 3 disulfides, C4-C17, C209-C248, and C211-C264. 16 C65 is buried in the hydrophobic core of the N-terminal domain and is therefore unpaired. 17,18 The other 2 Cys residues in GP Ib␣, C484 and C485, are located near the transmembrane (TM) domain and are conserved across species. The Ib␣/Ib␤ ratio of 1:1 in the receptor complex predicts that only 1 of the 2 Cys residues forms a disulfide bond with the membrane-proximal C122 in GP Ib␤ (Figure 1). However, it is not clear which one is paired with C122, and, more intriguingly, what role the free thiol group of the other Cys residue plays. It seems paradoxic that 2 neighboring Cys residues are exposed to presumably similar, if not the same, oxidizing environments, yet they have entirely different fates.In the present study, we sought to assign the disulfide between GP Ib␣ and GP Ib␤. To our surprise, we found that both C484 and C485 are linked to GP Ib␤ by disulfide bonds. In other words, contrary to the currently prevailing model, 1 Ib␣ subunit is linked through disulfide bonds to 2 Ib␤ subunits. Materials and methods Vectors and antibodiesThe vector pDX 19,20 was used in the expression of GP Ib-IX complex in Chinese hamster ovary (CHO) cells. The CHO K1 cell line was obtained from ATCC (Manassas, VA). The expression vector pGEX-4T-3 was purchased from Amersham Biosciences (Piscataway, NJ). WM23, an anti-Ib␣ monoclonal antibody, was kindly provided by Dr M. Berndt. Other antibodies against individual subunits of GP Ib-IX complex, including FMC25, Gi27, SZ2, and AK2, were p...

The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodynamic effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine-isoleucine-valine binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chemical denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the reduction in entropy of unfolding was typically several fold larger than the theoretical estimate of -4 cal K(-1) mol(-1) derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, respectively. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive.

Subunit “a” is associated with the membrane-bound (VO) complex of eukaryotic vacuolar H+-ATPase (V-ATPase) acidification machinery. It has also been shown recently to be involved in diverse membrane fusion/secretory functions independent of acidification. Here, we report the crystal structure of the N-terminal cytosolic domain from the Meiothermus ruber subunit “I” homolog of subunit a. The structure is composed of a curved long central α-helix bundle capped on both ends by two lobes with similar α/β architecture. Based on the structure, a reasonable model of its eukaryotic subunit a counterpart was obtained. The crystal structure and model fit well into reconstruction densities from electron microscopy of prokaryotic and eukaryotic V-ATPases, respectively, clarifying their orientations and interactions and revealing features that could enable subunit a to play a role in membrane fusion/secretion.

The calmodulin hypothesis of ectodomain shedding stipulates that calmodulin, an intracellular Ca2+-dependent regulatory protein, associates with the cytoplasmic domain of L-selectin to regulate ectodomain shedding of L-selectin on the other side of the plasma membrane. To understand the underlying molecular mechanism, we have characterized the interactions of calmodulin with two peptides derived from human L-selectin. The peptide ARR18 corresponds to the entire cytoplasmic domain of L-selectin (residues Ala317–Tyr334 in the mature protein), and CLS corresponds to residues Lys280–Tyr334, which contains the entire transmembrane and cytoplasmic domains of L-selectin. Monitoring the interaction by fluorescence spectroscopy and other biophysical techniques, we found that calmodulin can bind to ARR18 in aqueous solutions or the L-selectin cytoplasmic domain of CLS reconstituted in the phosphatidylcholine bilayer, both with an affinity of approximate 2 μM. The association is calcium-independent, dynamic and involves both lobes of calmodulin. In a phospholipid bilayer, the positively charged L-selectin cytoplasmic domain of CLS is associated with anionic phosphatidylserine lipids at the membrane interface through electrostatic interactions. Under conditions where the phosphatidylserine content mimics that in the inner leaflet of the cell plasma membrane, the interaction between calmodulin and CLS becomes undetectable. These results suggest that the association of calmodulin with L-selectin in the cell can be influenced by the membrane bilayer, and that anionic lipids may modulate ectodomain shedding of transmembrane receptors.

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Ca2+–Calmodulin binding to neuronal v-ATPase V0 subunit a1 (V100) regulates SNARE complex assembly for a putative subset of synaptic vesicles that sustain spontaneous release in Drosophila.

The fundamental processes of membrane fission and fusion determine size and copy numbers of intracellular organelles. While SNARE proteins and tethering complexes mediate intracellular membrane fusion, fission requires the presence of dynamin or dynamin-related proteins. Here we study these reactions in native yeast vacuoles and find that the yeast dynamin homolog Vps1 is not only an essential part of the fission machinery, but also controls membrane fusion by generating an active Qa SNARE- tethering complex pool, which is essential for trans-SNARE formation. Our findings provide new insight into the role of dynamins in membrane fusion by directly acting on SNARE proteins.

Chloroquine (CQ) and other quinoline-containing antimalarials are important drugs with many therapeutic benefits as well as adverse effects. However, the molecular targets underlying most such effects are largely unknown. By taking a novel functional genomics strategy, which employs a unique combination of genome-wide drug-gene synthetic lethality (DGSL), gene-gene synthetic lethality (GGSL), and dosage suppression (DS) screens in the model organism Saccharomyces cerevisiae and is thus termed SL/DS for simplicity, we found that CQ inhibits the thiamine transporters Thi7, Nrt1, and Thi72 in yeast. We first discovered a thi3Δ mutant as hypersensitive to CQ using a genome-wide DGSL analysis. Using genome-wide GGSL and DS screens, we then found that a thi7Δ mutation confers severe growth defect in the thi3Δ mutant and that THI7 overexpression suppresses CQ-hypersensitivity of this mutant. We subsequently showed that CQ inhibits the functions of Thi7 and its homologues Nrt1 and Thi72. In particular, the transporter activity of wild-type Thi7 but not a CQ-resistant mutant (Thi7T287N) was completely inhibited by the drug. Similar effects were also observed with other quinoline-containing antimalarials. In addition, CQ completely inhibited a human thiamine transporter (SLC19A3) expressed in yeast and significantly inhibited thiamine uptake in cultured human cell lines. Therefore, inhibition of thiamine uptake is a conserved mechanism of action of CQ. This study also demonstrated SL/DS as a uniquely effective methodology for discovering drug targets.