Dynamic oligopeptide acquisition by the RagAB transporter fromPorphyromonas gingivalis

Abstract

38Porphyromonas gingivalis, an asaccharolytic Bacteroidetes, is a keystone pathogen in human 39 periodontitis that may also contribute to the development of other chronic inflammatory diseases, 40 such as rheumatoid arthritis, cardiovascular disease and Alzheimer's disease. P. gingivalis utilizes 41 protease-generated peptides derived from extracellular proteins for growth, but how those peptides 42 enter the cell is not clear. Here we identify RagAB as the outer membrane importer for peptides. X-43 ray crystal structures show that the transporter forms a dimeric RagA 2 B 2 complex with the RagB 44 substrate binding surface-anchored lipoprotein forming a closed lid on the TonB-dependent 45 transporter RagA. Cryo-electron microscopy structures reveal the opening of the RagB lid and thus 46 provide direct evidence for a "pedal bin" nutrient uptake mechanism. Together with mutagenesis, 47 peptide binding studies and RagAB peptidomics, our work identifies RagAB as a dynamic OM 48 oligopeptide acquisition machine with considerable substrate selectivity that is essential for the 49 efficient utilisation of proteinaceous nutrients by P. gingivalis. 50 51 Introduction 52 The Gram-negative Bacteroidetes are abundant members of the human microbiota, especially in 53 the gut. Outside the gut, Bacteroidetes often cause disease, with the best-known examples being 54 the oral Bacteroidetes Porphyromonas gingivalis and Tannerella forsythia that are part of the "red 55 complex" involved in periodontitis 1 , the most prevalent infection-driven chronic inflammation in the 56 Western world 2 . Accumulating evidence suggests a link between periodontitis and other chronic 57 inflammatory diseases, including rheumatoid arthritis, Alzheimer's disease, chronic obstructive 58 pulmonary disease and cardiovascular disease 3-7 . Given this link, and the fastidious growth 59 requirements of P. gingivalis, it is important to understand how this key pathogen thrives and 60 causes dysbiosis of the oral microbiota in the biofilm on the tooth surface below the gum line, 61 leading to inflammation and periodontal tissue destruction. 63Unlike many human gut Bacteroidetes that specialise in degrading complex host and dietary 64 glycans, P. gingivalis is asaccharolytic and exclusively utilises peptides for growth 8 . Those 65 peptides are generated by multiple proteases, including several secreted endopeptidases and 66 periplasmic di-and tri-peptidyl-peptidases 9,10 . The best-known P. gingivalis endopeptidases are the 67 gingipains, large and abundant surface-anchored cysteine proteases with cumulative trypsin-like 68 activity, which are essential for P. gingivalis virulence and growth on proteins as the sole source of 69 carbon 11 . Crucially, it is not clear how gingipain-generated peptides are taken up by P. gingivalis. A 70 role in this process has been proposed for the RagAB outer membrane (OM) protein complex 12 , 71 which consists of a TonB dependent transporter (TBDT) RagA (PG_0185) and a substrate binding72 surface lipoprotein RagB (PG_0186...

The evolution of SPHIRE-crYOLO particle picking and its application in automated cryo-EM processing workflows

“…crYOLO [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] . For example, Pang et al 23 used crYOLO to selectively pick particles, which were attached to liposomes; Rogala et al 14 highlighted in their study about mTORC1 that crYOLO was especially useful to exclude particles on carbon; Joppe et al 24 made use of crYOLO in a streamlined pipeline for rapid structure determination of yeast fatty acid synthase; and the new filament mode was recently used by Pospich et al to examine the structural effects of toxins on actin filaments 16 .…”