Multivalent interactions make adherens junction–cytoskeletal linkage robust during morphogenesis

Embryogenesis requires cells to change shape and move without disrupting epithelial integrity. This requires robust, responsive linkage between adherens junctions and the actomyosin cytoskeleton. Using Drosophila morphogenesis, we define molecular mechanisms mediating junction–cytoskeletal linkage and explore the role of mechanosensing. We focus on the junction–cytoskeletal linker Canoe, a multidomain protein. We engineered the canoe locus to define how its domains mediate its mechanism of action. To our surprise, the PDZ and FAB domains, which we thought connected junctions and F-actin, are not required for viability or mechanosensitive recruitment to junctions under tension. The FAB domain stabilizes junctions experiencing elevated force, but in its absence, most cells recover, suggesting redundant interactions. In contrast, the Rap1-binding RA domains are critical for all Cno functions and enrichment at junctions under tension. This supports a model in which junctional robustness derives from a large protein network assembled via multivalent interactions, with proteins at network nodes and some node connections more critical than others.

Structural evidence for a proline-specific glycopeptide recognition domain in an O-glycopeptidase

The glycosylation of proteins is typically considered as a stabilizing modification, including resistance to proteolysis. A class of peptidases, referred to as glycopeptidases or O-glycopeptidases, circumvent the protective effect of glycans against proteolysis by accommodating the glycans in their active sites as specific features of substrate recognition. IMPa from Pseudomonas aeruginosa is such an O-glycopeptidase that cleaves the peptide bond immediately preceding a site of O-glycosylation, and through this glycoprotein-degrading function contributes to the host-pathogen interaction. IMPa, however, is a relatively large multidomain protein and how its additional domains may contribute to its function remains unknown. Here, through the determination of a crystal structure of IMPa in complex with an O-glycopeptide, we reveal that the N-terminal domain of IMPa, which is classified in Pfam as IMPa_N_2, is a proline recognition domain that also shows the properties of recognizing an O-linked glycan on the serine/threonine residue following the proline. The proline is bound in the center of a bowl formed by four functionally conserved aromatic amino acid side chains while the glycan wraps around one of the tyrosine residues in the bowl to make classic aromatic ring-carbohydrate CH-π interactions. This structural evidence provides unprecedented insight into how the ancillary domains in glycoprotein-specific peptidases can noncatalytically recognize specific glycosylated motifs that are common in mucin and mucin-like molecules.

Observing the nonvectorial yet cotranslational folding of a multidomain protein, LDL receptor, in the ER of mammalian cells

Proteins have evolved by incorporating several structural units within a single polypeptide. As a result, multidomain proteins constitute a large fraction of all proteomes. Their domains often fold to their native structures individually and vectorially as each domain emerges from the ribosome or the protein translocation channel, leading to the decreased risk of interdomain misfolding. However, some multidomain proteins fold in the endoplasmic reticulum (ER) nonvectorially via intermediates with nonnative disulfide bonds, which were believed to be shuffled to native ones slowly after synthesis. Yet, the mechanism by which they fold nonvectorially remains unclear. Using two-dimensional (2D) gel electrophoresis and a conformation-specific antibody that recognizes a correctly folded domain, we show here that shuffling of nonnative disulfide bonds to native ones in the most N-terminal region of LDL receptor (LDLR) started at a specific timing during synthesis. Deletion analysis identified a region on LDLR that assisted with disulfide shuffling in the upstream domain, thereby promoting its cotranslational folding. Thus, a plasma membrane-bound multidomain protein has evolved a sequence that promotes the nonvectorial folding of its upstream domains. These findings demonstrate that nonvectorial folding of a multidomain protein in the ER of mammalian cells is more coordinated and elaborated than previously thought. Thus, our findings alter our current view of how a multidomain protein folds nonvectorially in the ER of living cells.

LAMPA, LArge Multidomain Protein Annotator, and its application to RNA virus polyproteins

Motivation To facilitate accurate estimation of statistical significance of sequence similarity in profile–profile searches, queries should ideally correspond to protein domains. For multidomain proteins, using domains as queries depends on delineation of domain borders, which may be unknown. Thus, proteins are commonly used as queries that complicate establishing homology for similarities close to cutoff levels of statistical significance. Results In this article, we describe an iterative approach, called LAMPA, LArge Multidomain Protein Annotator, that resolves the above conundrum by gradual expansion of hit coverage of multidomain proteins through re-evaluating statistical significance of hit similarity using ever smaller queries defined at each iteration. LAMPA employs TMHMM and HHsearch for recognition of transmembrane regions and homology, respectively. We used Pfam database for annotating 2985 multidomain proteins (polyproteins) composed of >1000 amino acid residues, which dominate proteomes of RNA viruses. Under strict cutoffs, LAMPA outperformed HHsearch-mediated runs using intact polyproteins as queries by three measures: number of and coverage by identified homologous regions, and number of hit Pfam profiles. Compared to HHsearch, LAMPA identified 507 extra homologous regions in 14.4% of polyproteins. This Pfam-based annotation of RNA virus polyproteins by LAMPA was also superior to RefSeq expert annotation by two measures, region number and annotated length, for 69.3% of RNA virus polyprotein entries. We rationalized the obtained results based on dependencies of HHsearch hit statistical significance for local alignment similarity score from lengths and diversities of query-target pairs in computational experiments. Availability and implementation LAMPA 1.0.0 R package is placed at github (https://github.com/Gorbalenya-Lab/LAMPA). Supplementary information Supplementary data are available at Bioinformatics online.