James D Lear's research while affiliated with University of Pennsylvania and other places

The influenza A/M2 protein is a homotetrameric single-pass integral membrane protein encoded by the influenza A viral genome. Its transmembrane domain represents both a crucial drug target and a minimalistic model system for transmembrane proton transport and charge stabilization. Recent structural and functional studies of M2 have suggested that the proton transport mechanism involves sequential extraviral protonation and intraviral deprotonation of a highly conserved His37 side chain by the transported proton, consistent with a pH-activated proton shuttle mechanism. Multiple tautomeric forms of His can be formed, and it is not known whether they contribute to the mechanism of proton shuttling. Here we present the thermodynamic and functional characterization of an unnatural amino acid mutant at His37, where the imidazole side chain is substituted with a 4-thiazolyl group that is unable to undergo tautomerization and has a significantly lower solution pKa. The mutant construct has a similar stability to the wild-type protein at pH8 in bilayers and is virtually inactive at external pH7.4 in a semiquantitative liposome flux assay as expected from its lower sidechain pKa. However when the external buffer pH is lowered to 4.9 and 2.4, the mutant shows increasing amantadine sensitive flux of a similar magnitude to that of the wild type construct at pH7.4 and 4.9 respectively. These findings are in line with mechanistic hypotheses suggesting that proton flux through M2 is mediated by proton exchange from adjacent water molecules with the His37 sidechain, and that tautomerization is not required for proton translocation. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.

Dimerization and temperature dependence of IC-p150Glued interaction. (A/B) Oligomeric state and association with IC1–124 was examined as a function of temperature for both CC1B and CC1. A single speed of 25000 rpm was analyzed at 5, 10, 15, 20 and 25°C. The dimerization of CC1B and association with IC1–124 is temperature dependent (A), while no change in either dimerization or association is seen for CC1 (B). IC1–124 is monomeric at all temperatures. (TIF)

Alignment of dynein IC and p150Glued sequences from different organisms and predicted coiled-coil regions. (TIF)

Chi square analysis of p150Glued oligomer (A) and IC-p150Glued complex (B) in salt dependence assays. (TIF)

Chi square analysis of p150Glued oligomer formation and IC-p150Glued complex formation. (A) The radial absorbance of the CC1, CC1A and CC1B constructs was fit to a monomer-dimer model affording an optimal dissociation constant. Next, the dissociation constant was fixed at different values centered about the best-fit value and the resulting chi squared value was determined. The chi squared was plotted against the fixed dissociation constants. A sharp rise in the chi squared value indicates limiting values. For instance, the lower limit of the dissociation constant is 8 for CC1(A, left panel). However, the value may be much greater. (B) The radial absorbance of IC1–124 mixed with CC1, CC1A and CC1B was fit to a 2IC +CC ⇔ (IC)2(CC)1 model affording an optimal dissociation constant. The same chi square analysis was perfomed. Note that CC1B + IC1–124 is well constrained at 12. (B) The IC-p150Glued complex data was fit by fixing the dissociation constant around the best-fit value and the chi squared value was recorded. (TIF)

Thermal Denaturation of p150Glued fragments with IC1–44. (A) CC1A alone (diamonds) or incubated with an equimolar concentration of IC1–44 (squares). (B) CC1B alone (diamonds) or incubated with an equimolar concentration of IC1–44 (squares). (TIF)

Coiled-coil and isoelectric point sequence analysis of IC and p150Glued binding regions. (A/B) Both the intermediate chain and p150Glued contain regions that have a propensity to exist in a coiled-coil, denoted by a P-score of 0.025 or less (1). Coiled-coil prediction programs indicate a possible N-terminal coiled-coil spanning residues 1–44 of the IC (A). In addition, sequence alignment (2) and coiled-coil prediction of p150Glued indicate a conserved break in the coiled-coil region. Based on this break we designed two new fragments denoted CC1A and CC1B (B). (C/D) The average isoelectric point of the intermediate chain, residues 1–151 and p150Glued CC1 was determined by calculating the isoelectric point for 28 residues, every 7 residues. Both the p150Glued and IC binding sites are highlighted with a grey box. Note that the isoelectric point of the IC1–44 is 9.7, while p150Glued (415–530) is 4.43, indicating the interaction may primarily be governed by electrostatic interactions. (TIF)

p150Glued CC1 is a parallel coiled-coil. (A) Upon incubation of Cys-CC1 with 3,5-DMBA we see the presence of a band equivalent to two times the molecular weight of Cys-CC1 (arrow). This indicates that the two cysteines are in close proximity in the CC1 dimer and are able to be crosslinked. (TIF)

Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC) and the dynactin p150(Glued); however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10-44, is sufficient for binding p150(Glued). Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150(Glued) form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150(Glued) fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD) experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150(Glued). Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.