We have observed a common sequence motif in membrane proteins, which we call a glycine zipper. Glycine zipper motifs are strongly overrepresented and conserved in membrane protein sequences, and mutations in glycine zipper motifs are deleterious to function in many cases. The glycine zipper has a significant structural impact, engendering a strong driving force for right-handed packing against a neighboring helix. Thus, the presence of a glycine zipper motif leads directly to testable structural hypotheses, particularly for a subclass of glycine zipper proteins that form channels. For example, we suggest that the membrane pores formed by the amyloid- peptide in vitro are constructed by glycine zipper packing and find that mutations in the glycine zipper motif block channel formation. Our findings highlight an important structural motif in a wide variety of normal and pathological processes.amyloid- ͉ membrane channel ͉ membrane protein structure ͉ prion ͉ transmembrane helix M ore than 13 structures a day are currently being deposited in the Protein Data Bank (1), and structural genomics centers have been created to obtain structures even faster [such as the National Institute of General Medical Sciences (NIGMS) Protein Structure Initiative, www.nigms.nih.gov͞psi]. In this assault on protein structure, however, technical challenges have left membrane proteins far behind. Membrane protein structures are currently being solved at Ϸ0.2% of the pace of soluble proteins (2). Thus, membrane protein biochemists are relatively starved for structural insight into these key proteins. In the absence of dramatic technical improvements, alternatives to experimental structure determination are needed.Here, we describe a transmembrane (TM) sequence motif, the glycine zipper, that can lead directly to structural models for many membrane proteins. The most significant glycine zipper sequence patterns are (G,A,S)XXXGXXXG and GXXXGX-XX(G,S,T). These patterns contain a GXXXG motif, which is known to be important in TM helix homodimers where the Gly faces are in direct contact (3-5). The GXXXG sequence pattern is statistically overrepresented in membrane proteins in general, not just in TM homodimers (4). Nevertheless, the structural role of the GXXXG pattern in other types of TM helix packing interactions has not been elucidated. We find that the addition of an appropriately spaced small residue, as found in the glycine zipper, leads to a distinct preference for right-handed packing against a heterologous helix surface. Thus, the presence of a glycine zipper generates a strong helix packing prediction, particularly for homooligomeric channel proteins, providing a structural foundation for hypothesis-driven investigations. MethodsGlycine Zipper Motif Search. We started with Swiss-Prot release 41.15 containing 129,996 proteins (6). All sequences Ͻ50 residues in length were removed, leaving 125,887 proteins. Helical membrane proteins were identified by using the Eisenberg hydrophobicity scale and a window length of 21 residues (7). ...
Understanding the energetics of molecular interactions is fundamental to all of the central quests of structural biology including structure prediction and design, mapping evolutionary pathways, learning how mutations cause disease, drug design, and relating structure to function. Hydrogen-bonding is widely regarded as an important force in a membrane environment because of the low dielectric constant of membranes and a lack of competition from water. Indeed, polar residue substitutions are the most common disease-causing mutations in membrane proteins. Because of limited structural information and technical challenges, however, there have been few quantitative tests of hydrogen-bond strength in the context of large membrane proteins. Here we show, by using a double-mutant cycle analysis, that the average contribution of eight interhelical side-chain hydrogen-bonding interactions throughout bacteriorhodopsin is only 0.6 kcal mol(-1). In agreement with these experiments, we find that 4% of polar atoms in the non-polar core regions of membrane proteins have no hydrogen-bond partner and the lengths of buried hydrogen bonds in soluble proteins and membrane protein transmembrane regions are statistically identical. Our results indicate that most hydrogen-bond interactions in membrane proteins are only modestly stabilizing. Weak hydrogen-bonding should be reflected in considerations of membrane protein folding, dynamics, design, evolution and function.
One of the hallmarks of membrane protein structure is the high frequency of transmembrane helix kinks, which commonly occur at proline residues. Because the proline side chain usually precludes normal helix geometry, it is reasonable to expect that proline residues generate these kinks. We observe, however, that the three prolines in bacteriorhodopsin transmembrane helices can be changed to alanine with little structural consequences. This finding leads to a conundrum: if proline is not required for helix bending, why are prolines commonly present at bends in transmembrane helices? We propose an evolutionary hypothesis in which a mutation to proline initially induces the kink. The resulting packing defects are later repaired by further mutation, thereby locking the kink in the structure. Thus, most prolines in extant proteins can be removed without major structural consequences. We further propose that nonproline kinks are places where vestigial prolines were later removed during evolution. Consistent with this hypothesis, at 14 of 17 nonproline kinks in membrane proteins of known structure, we find prolines in homologous sequences. Our analysis allows us to predict kink positions with >90% reliability. Kink prediction indicates that different G protein-coupled receptor proteins have different kink patterns and therefore different structures.
Somatic mosaicism in genetic disease generally results from a de novo deleterious mutation during embryogenesis. We now describe a somatic mosaicism due to the unusual mechanism of in vivo reversion to normal of an inherited mutation. The propositus was an adenosine deaminase-deficient (ADA-) child with progressive clinical improvement and unexpectedly mild biochemical and immunologic abnormalities. Mosaicism due to reversion was evidenced by absence of a maternally transmitted deleterious mutation in 13/15 authenticated B cell lines and in 17% of single alleles cloned from blood DNA, despite retention of a maternal 'private' ADA polymorphism linked to the mutation. Establishment of significant somatic mosaicism following reversion to normal could modify any disorder in which revertant cells have a selective advantage.
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