The C␣OH⅐⅐⅐O hydrogen bond has been given little attention as a determinant of transmembrane helix association. Stimulated by recent calculations suggesting that such bonds can be much stronger than has been supposed, we have analyzed 11 known membrane protein structures and found that apparent carbon ␣ hydrogen bonds cluster frequently at glycine-, serine-, and threonine-rich packing interfaces between transmembrane helices. Parallel righthanded helix-helix interactions appear to favor C␣OH⅐⅐⅐O bond formation. In particular, C␣OH⅐⅐⅐O interactions are frequent between helices having the structural motif of the glycophorin A dimer and the GxxxG pair. We suggest that C␣OH⅐⅐⅐O hydrogen bonds are important determinants of stability and, depending on packing, specificity in membrane protein folding.T he hydrogen bond is a key element in the interplay between stability and specificity in protein folding. The desolvation penalty associated with burial of polar side chains in an aqueous environment is not always fully recovered by hydrogen bond formation, so hydrogen bonds provide a small or even unfavorable net energy contribution to folding. However, the strength and directionality of hydrogen bonds make them an important factor in discriminating between correctly folded and misfolded states. Hence, polar interactions tend to contribute more to specificity than to stability in soluble proteins (1-3). Conversely, in the apolar environment of biological membranes donor and acceptor groups cannot be satisfied by the solvent, and hydrogen bonds strongly stabilize the helical conformation of membrane spanning domains (4) and can stabilize tertiary interactions as well (5-9). We are interested in the role of hydrogen bonds in the association of transmembrane helices, a stage that is pivotal in the folding of membrane proteins (4). Recently, the DeGrado group and our laboratory showed that the substitution of a single polar amino acid residue into model transmembrane helices induces homooligomerization (10 -13); the association driven by hydrogen bonding can be strong and independent of packing details. Thus, in the apolar environment, the strength of hydrogen bonds can stabilize the association of transmembrane helices, although a lack of a need for sequence specificity could create a danger of inducing promiscuous association (10, 13).Weaker hydrogen bonds, such as those between carbon and oxygen atoms (COH⅐⅐⅐O), have received little attention in the membrane protein field, and their occurrence in membrane proteins has never been surveyed. The C␣ is an activated carbon donor because it is bound to the electron-withdrawing amide NOH and CAO groups, and, in soluble proteins, hydrogen bonds between main-chain C␣OH groups and backbone or side-chain oxygen atoms are often observed (14-17). Despite its abundance, the structural contribution of the C␣OH⅐⅐⅐O hydrogen bond has been unclear and its interaction energy has been believed to be small. Recently, by using ab initio calculations, Vargas et al. (18) and Scheiner et al. (19...

A biological membrane is conceptualized as a system in which membrane proteins are naturally matched to the equilibrium thickness of the lipid bilayer. Cholesterol, in addition to lipid composition, has been suggested to be a major regulator of bilayer thickness in vivo because measurements in vitro have shown that cholesterol can increase the thickness of simple phospholipid͞cholesterol bilayers. Using solution x-ray scattering, we have directly measured the average bilayer thickness of exocytic pathway membranes, which contain increasing amounts of cholesterol. The bilayer thickness of membranes of the endoplasmic reticulum, the Golgi, and the basolateral and apical plasma membranes, purified from rat hepatocytes, were determined to be 37.5 ؎ 0.4 Å, 39.5 ؎ 0.4 Å, 35.6 ؎ 0.6 Å, and 42.5 ؎ 0.3 Å, respectively. After cholesterol depletion using cyclodextrins, Golgi and apical plasma membranes retained their respective bilayer thicknesses whereas the bilayer thickness of the endoplasmic reticulum and the basolateral plasma membrane decreased by 1.0 Å. Because cholesterol was shown to have a marginal effect on the thickness of these membranes, we measured whether membrane proteins could modulate thickness. Protein-depleted membranes demonstrated changes in thickness of up to 5 Å, suggesting that (i) membrane proteins rather than cholesterol modulate the average bilayer thickness of eukaryotic cell membranes, and (ii) proteins and lipids are not naturally hydrophobically matched in some biological membranes. A marked effect of membrane proteins on the thickness of Escherichia coli cytoplasmic membranes, which do not contain cholesterol, was also observed, emphasizing the generality of our findings.

Crohn’s disease (CD), a form of inflammatory bowel disease, has a higher prevalence in Ashkenazi Jewish than in non-Jewish European populations. To define the role of non-synonymous mutations, we performed exome sequencing of Ashkenazi Jewish patients with CD, followed by array-based genotyping and association analysis in 2,066 CD cases and 3,633 healthy controls. We detected association signals in the LRRK2 gene that conferred CD risk (N2081D variant, P=9.5×10−10) or protection (N551K variant, tagging R1398H-associated haplotype, P=3.3×10−8). These variants affected CD age of onset, disease location, LRRK2 activity, and autophagy. Bayesian network analysis of CD patient intestinal tissue further implicated LRRK2 in CD pathogenesis. Analysis of the extended LRRK2 locus in 24,570 CD cases, patients with Parkinson’s disease (PD), and healthy controls revealed extensive pleiotropy, with similar genetic effects between CD and PD in both Ashkenazi Jewish and non-Jewish cohorts. The LRRK2 N2081D CD risk allele is located in the same kinase domain as G2019S, a mutation that is the major genetic cause of familial and sporadic PD. Like the G2019S mutation, the N2081D variant is associated with increased kinase activity, whereas neither N551K nor R1398H on the protective haplotype altered kinase activity. R1398H, but not N551K, increased GTPase activity, thereby deactivating LRRK2. The presence of shared LRRK2 alleles in CD and PD provides refined insight into disease mechanisms and may have major implications for the treatment of these two seemingly unrelated diseases.

The small multidrug resistance family of transporters is widespread in bacteria and is responsible for bacterial resistance to toxic aromatic cations by proton-linked ef¯ux. We have determined the threedimensional (3D) structure of the Escherichia coli multidrug transporter EmrE by electron cryomicroscopy of 2D crystals, including data to 7.0 A Ê resolution. The structure of EmrE consists of a bundle of eight transmembrane a-helices with one substrate molecule bound near the centre. The substrate binding chamber is formed from six helices and is accessible both from the aqueous phase and laterally from the lipid bilayer. The most remarkable feature of the structure of EmrE is that it is an asymmetric homodimer. The possible arrangement of the two polypeptides in the EmrE dimer is discussed based on the 3D density map.