Unusual Armadillo Fold in the Human General Vesicular Transport Factor p115

The golgin family gives identity and structure to the Golgi apparatus and is part of a complex protein network at the Golgi membrane. The golgin p115 is targeted by the GTPase Rab1a, contains a large globular head region and a long region of coiled-coil which forms an extended rod-like structure. p115 serves as vesicle tethering factor and plays an important role at different steps of vesicular transport. Here we present the 2.2 Å-resolution X-ray structure of the globular head region of p115. The structure exhibits an armadillo fold that is decorated by elongated loops and carries a C-terminal non-canonical repeat. This terminal repeat folds into the armadillo superhelical groove and allows homodimeric association with important implications for p115 mediated multiple protein interactions and tethering

Armadillo Motifs Involved in Vesicular Transport

Armadillo (ARM) repeat proteins function in various cellular processes including vesicular transport and membrane tethering. They contain an imperfect repeating sequence motif that forms a conserved three-dimensional structure. Recently, structural and functional insight into tethering mediated by the ARM-repeat protein p115 has been provided. Here we describe the p115 ARM-motifs for reasons of clarity and nomenclature and show that both sequence and structure are highly conserved among ARM-repeat proteins. We argue that there is no need to invoke repeat types other than ARM repeats for a proper description of the structure of the p115 globular head region. Additionally, we propose to define a new subfamily of ARM-like proteins and show lack of evidence that the ARM motifs found in p115 are present in other long coiled-coil tethering factors of the golgin family

A protein functional leap: how a single mutation reverses the function of the transcription regulator TetR

Today's proteome is the result of innumerous gene duplication, mutagenesis, drift and selection processes. Whereas random mutagenesis introduces predominantly only gradual changes in protein function, a case can be made that an abrupt switch in function caused by single amino acid substitutions will not only considerably further evolution but might constitute a prerequisite for the appearance of novel functionalities for which no promiscuous protein intermediates can be envisaged. Recently, tetracycline repressor (TetR) variants were identified in which binding of tetracycline triggers the repressor to associate with and not to dissociate from the operator DNA as in wild-type TetR. We investigated the origin of this activity reversal by limited proteolysis, CD spectroscopy and X-ray crystallography. We show that the TetR mutant Leu17Gly switches its function via a disorder–order mechanism that differs completely from the allosteric mechanism of wild-type TetR. Our study emphasizes how single point mutations can engender unexpected leaps in protein function thus enabling the appearance of new functionalities in proteins without the need for promiscuous intermediates

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Electrostatic surfaces comparison of the superhelical grooves of the p115^GHR, β-catenin and karyopherin-α.

<p>Blue indicates positive charge and red negative charge at the level of 10 kT/e. The amino terminus of the molecule is at the top of the figure.</p

Schematic overview of full-length p115.

<p>The construct comprising p115^GHR used for crystallization is shown in gray.</p

Interaction of p115^GHR and the COG complex subunit COG2.

<p>(A) Electrostatic surfaces of p115^GHR. The amino terminus of the molecule is at the top of the figure. Blue indicates positive charge and red negative charge at the level of 10 kT/e. (B) Conserved residues of p115 and the yeast homolog Uso1p form a highly charged surface of exposed helices which define the COG2 binding site.</p

Crystal structure of p115^GHR.

<p>(A) Stereo view of the overall structure. The protein is composed of 11 armadillo repeats. (B) Structure-based sequence alignment of the p115 armadillo repeats and the loop regions. Consensus residues that define the conserved hydrophobic residues of each armadillo repeat are highlighted in blue; amino acids that define conserved polar, neutral residues are highlighted in green. Glycine and proline residues are highlighted in brown and olive, respectively. The repeat numbers are shown on the left. The sequences that form helices H1, H2, and H3 are indicated as green, blue and yellow cylinders. The corresponding ARM loops are marked on top, the USO helix is indicated as red cylinder. (C) N-terminal armadillo-like helical domain (left) and C-terminal Uso1 head domain (right) of p115^GHR, elongated loops with at least 5 residues are colored in red (H1 green, H2 blue, H3 yellow).</p

Dimeric arrangement of the p115 globular head region.

<p>(A) <i>B</i>-factor representation (“S-“ and “W-view”) of p115^GHR molecules aligned by crystal symmetry. The intermolecular contact area (blue) is among the most rigid parts of the structure. (B) Depending on the orientation, the crystallographic dimer of p115^GHR has a single-head (“O-view”) or double-lobed globular appearance (“W-” and ”V-view”).</p