The relationship between water bridges and the polyproline II conformation: a large-scale analysis of molecular dynamics simulations and crystal structures.

It has been suggested that denatured proteins are predisposed toward the left-handed polyproline II (P(II)) conformation. One possible source of P(II) stability in the denatured state is water bridges. Water bridges are networks of water molecules that link nearby hydrogen bond acceptors and/or donors on proteins. On the basis of the proposed behavior of P(II) and water bridges, the propensity of a residue to participate in water bridges should be correlated with its P(II) propensity. To test this hypothesis, we analyzed the following data sets: 2351 high-resolution crystal structures, and the native and denatured states of 188 different proteins from all-atom, explicit-solvent molecular dynamics (MD) simulations, which are part of our Dynameomics effort. We found that water bridges do not explain the high frequency of P(II) in denatured states; such bridges are less frequent around P(II) than around other conformations. Thus, this analysis casts doubt on water bridges as a dominant factor determining the residue-based P(II) propensities.

Mutagenesis data in the automated prediction of transmembrane helix dimers.

We present a molecular modeling protocol that selects modeled protein structures based on experimental mutagenesis results. The computed effect of a point mutation should be consistent with its experimental effect for correct models; mutations that do not affect protein stability and function should not affect the computed energy of a correct model while destabilizing mutations should have unfavorable computed energies. On the other hand, an incorrect model will likely display computed energies that are inconsistent with experimental results. We added terms to our energy function which penalize models that are inconsistent with experimental results. This creates a selective advantage for models that are consistent with experimental results in the Monte Carlo simulated annealing protocol we use to search conformational space. We calibrated our protocol to predict the structure of transmembrane helix dimers using glycophorin A as a model system. Inclusion of mutational data in this protocol compensates for the limitations of our force field and the limitations of our conformational search. We demonstrate an application of this structure prediction protocol by modeling the transmembrane region of the BNIP3 apoptosis factor.

E(z), a depth-dependent potential for assessing the energies of insertion of amino acid side-chains into membranes: derivation and applications to determining the orientation of transmembrane and interfacial helices.

We have developed an empirical residue-based potential (E(z) potential) for protein insertion in lipid membranes. Propensities for occurrence as a function of depth in the bilayer were calculated for the individual amino acid types from their distribution in known structures of helical membrane proteins. The propensities were then fit to continuous curves and converted to a potential using a reverse-Boltzman relationship. The E(z) potential demonstrated a good correlation with experimental data such as amino acid transfer free energy scales (water to membrane center and water to interface), and it incorporates transmembrane helices of varying composition in the membrane with trends similar to those obtained with translocon-mediated insertion experiments. The potential has a variety of applications in the analysis of natural membrane proteins as well as in the design of new ones. It can help in calculating the propensity of single helices to insert in the bilayer and estimate their tilt angle with respect to the bilayer normal. It can be utilized to discriminate amphiphilic helices that assume a parallel orientation at the membrane interface, such as those of membrane-active peptides. In membrane protein design applications, the potential allows an environment-dependent selection of amino acid identities.

A push-pull mechanism for regulating integrin function.

Homomeric and heteromeric interactions between the alphaIIb and beta3 transmembrane domains are involved in the regulation of integrin alphaIIbbeta3 function. These domains appear to interact in the inactivated state but separate upon integrin activation. Moreover, homomeric interactions may increase the level of alphaIIbbeta3 activity by competing for the heteromeric interaction that specifies the resting state. To test this model, a series of mutants were examined that had been shown previously to either enhance or disrupt the homomeric association of the alphaIIb transmembrane domain. One mutation that enhanced the dimerization of the alphaIIb transmembrane domain indeed induced constitutive alphaIIbbeta3 activation. However, a series of mutations that disrupted homodimerization also led to alphaIIbbeta3 activation. These results suggest that the homo- and heterodimerization motifs overlap in the alphaIIb transmembrane domain, and that mutations that disrupt the alphaIIb/beta3 transmembrane domain heterodimer are sufficient to activate the integrin. The data also imply a mechanism for alphaIIbbeta3 regulation in which the integrin can be shifted from its inactive to its active state by destabilizing an alphaIIb/beta3 transmembrane domain heterodimer and by stabilizing the resulting alphaIIb and beta3 transmembrane domain homodimers.

Dimerization of the transmembrane domain of Integrin alphaIIb subunit in cell membranes.

Homo- and hetero-oligomeric interactions between the transmembrane (TM) helices of integrin alpha and beta subunits may play an important role in integrin activation and clustering. As a first step to understanding these interactions, we used the TOXCAT assay to measure oligomerization of the wild-type alpha(IIb) TM helix and single-site TM domain mutants. TOXCAT measures the oligomerization of a chimeric protein containing a TM helix in the Escherichia coli inner membrane via the transcriptional activation of the gene for chloramphenicol acetyltransferase. We found the amount of chloramphenicol acetyltransferase induced by the wild-type alpha(IIb) TM helix was approximately half that induced by the strongly dimerizing TM helix of glycophorin A, confirming that the alpha(IIb) TM domain oligomerizes in biological membranes. Mutating each of the alpha(IIb) TM domain residues to either Ala, Leu, Ile, or Val revealed that a GXXXG motif mediates oligomerization. Further, we found that the residue preceding each glycine contributed to the oligomerization interface, as did the residue at position i + 4 after the second Gly of GXXXG. Thus, the sequence XXVGXXGGXXXLXX is critical for oligomerization of alpha(IIb) TM helix. These data were used to generate an atomic model of the alpha(IIb) homodimer, revealing a family of structures with right-handed crossing angles of 40 degrees to 60 degrees, consistent with a 4.0-residue periodicity, and with an interface rotated by 50 degrees relative to glycophorin A. Thus, although the alpha(IIb) TM helix makes use of the GXXXG framework, neighboring residues have evolved to engineer its dimerization interface, enabling it to subserve specific and specialized functions.