Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase

The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains, rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C-terminus through the central pore of the hexameric double-ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.

Evaluation of mass spectrometry MS/MS spectra for the presence of isopeptide crosslinked peptides

Isopeptide crosslinked proteins can be the product of transglutaminase or of exposure to organophosphorus toxicants (OP). Transglutaminase links glutamine to lysine with loss of ammonia. OP toxicants induce a link between glutamic acid and lysine with loss of water. Our goal was to establish criteria to distinguish real from false isopeptide crosslinks reported by software searches of mass spectrometry data. We used fragmentation spectra of tryptic peptides from MAP-rich tubulin Sus scrofa as a test system for detection of naturally-occurring isopeptide crosslinks. Data were analyzed with Protein Prospector. Criteria for the assignments included the presence of at least 1 crosslink specific product ion, fragment ions from both peptides, Protein Prospector scores ≥20, and best fit of the MS/MS data to the crosslinked peptide as opposed to a linear peptide. Out of 301,364 spectra, 15 potential transglutaminase-type crosslinked peptide candidates were identified. Manual evaluation of these MS/MS spectra reduced the number to 1 valid crosslink between Q112 of NFH and K368 of Tau. Immunopurification with anti-isopeptide 81D1C2 confirmed that MAP-rich tubulin contained only one isopeptide. Support for this isopeptide bond was obtained by showing that transglutaminase was capable of incorporating dansyl-aminohexyl -QQIV into K368. A model of the KIETHK-QLEAHNR isopeptide was synthesized with the aid of transglutaminase. MS/MS spectra of the model validated our interpretation of the native isopeptide. An OP-induced isopeptide bond between K163 of tubulin alpha-1A and E158 of tubulin beta-4B was induced by treating MAP-rich tubulin with 100 μM chlorpyrifos oxon. This crosslink was supported by the criteria described above and by the presence of diethoxyphospho-lysine 163 in the tubulin alpha-1A peptide. The information obtained in this work is valuable for future studies that aim to understand why exposure to OP is associated with increased risk of neurodegenerative disease.

Sortase-assembled pili in Corynebacterium diphtheriae are built using a latch mechanism

Gram-positive bacteria assemble pili (fimbriae) on their surfaces to adhere to host tissues and to promote polymicrobial interactions. These hair-like structures, although very thin (1 to 5 nm), exhibit impressive tensile strengths because their protein components (pilins) are covalently crosslinked together via lysine–isopeptide bonds by pilus-specific sortase enzymes. While atomic structures of isolated pilins have been determined, how they are joined together by sortases and how these interpilin crosslinks stabilize pilus structure are poorly understood. Using a reconstituted pilus assembly system and hybrid structural biology methods, we elucidated the solution structure and dynamics of the crosslinked interface that is repeated to build the prototypical SpaA pilus from Corynebacterium diphtheriae. We show that sortase-catalyzed introduction of a K190-T494 isopeptide bond between adjacent SpaA pilins causes them to form a rigid interface in which the LPLTG sorting signal is inserted into a large binding groove. Cellular and quantitative kinetic measurements of the crosslinking reaction shed light onto the mechanism of pilus biogenesis. We propose that the pilus-specific sortase in C. diphtheriae uses a latch mechanism to select K190 on SpaA for crosslinking in which the sorting signal is partially transferred from the enzyme to a binding groove in SpaA in order to facilitate catalysis. This process is facilitated by a conserved loop in SpaA, which after crosslinking forms a stabilizing latch that covers the K190-T494 isopeptide bond. General features of the structure and sortase-catalyzed assembly mechanism of the SpaA pilus are likely conserved in Gram-positive bacteria.

A Mycobacterium tuberculosis effector protein attacks host innate immunity by acting as an unusual ubiquitinating enzyme

Protein kinase G (PknG), a eukaryotic type serine-threonine protein kinase (STPK) in Mycobacterium tuberculosis (Mtb), is secreted into the cytosol of infected macrophages to promote intracellular survival of mycobacteria and has been considered as a promising therapeutic target for tuberculosis (TB) treatment. However, the molecular details of Mtb PknG-host intracellular interactions remain obscure. Here, we demonstrate that PknG serves as both the ubiquitin-activating enzyme (E1) and the ubiquitin ligase (E3) to promote ubiquitination and degradation of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TGF–β-activated kinase 1 (TAK1), and thus inhibits the NF-κB-mediated host innate immune responses. Surprisingly, PknG promotes the attachment of ubiquitin (Ub) to ubiquitin-conjugating enzyme (E2) UbcH7 via an isopeptide bond (UbcH7 K82-Ub), instead of a usual C86-Ub thiol-ester bond, and then promotes the discharge of Ub from UbcH7 by acting as an isopeptidase before attaching Ub to its substrates TRAF2 and TAK1. These results demonstrate that Mtb PknG promotes ubiquitination of the key components of the host innate immunity by acting as an unusual ubiquitinating enzyme to suppress innate immunity. Our findings provide a potential TB treatment via targeting unconventional ubiquitinating activities of PknG.SignificanceMycobacterium tuberculosis (Mtb) protein kinase G (PknG), which is critical for Mtb intracellular survival, is a promising target for tuberculosis (TB) treatment. However, the molecular mechanisms underlying PknG-host interactions remain largely unclear. Here we demonstrate that PknG serves as both the ubiquitin-activating enzyme and the ubiquitin ligase to promote the ubiquitination and degradation of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TGF-β-activated kinase 1 (TAK1), thus inhibiting NF-κB signaling activation. PknG promotes the attachment of ubiquitin to ubiquitin-conjugating enzyme UbcH7 via an isopeptide bond, instead of a usual thiol-ester bond, and releases the ubiquitin from UbcH7 by acting as an isopeptidase. These findings provide important information for rational development of TB treatment via targeting unconventional ubiquitinating activity of PknG.

The insufficiency of ATG4A in macroautophagy

During autophagy, LC3 and GABARAP proteins become covalently attached to phosphatidylethanolamine on the growing autophagosome. This attachment is also reversible. Deconjugation (or delipidation) involves the proteolytic cleavage of an isopeptide bond between LC3 or GABARAP and the phosphatidylethanolamine headgroup. This cleavage is carried about by the ATG4 family of proteases (ATG4A, B, C, and D). Many studies have established that ATG4B is the most active of these proteases and is sufficient for autophagy progression in simple cells. Here we examined the second most active protease, ATG4A, to map out key regulatory motifs on the protein and to establish its activity in cells. We utilized fully in vitro reconstitution systems in which we controlled the attachment of LC3/GABARAP members and discovered a role for a C-terminal LC3-interacting region on ATG4A in regulating its access to LC3/GABARAP. We then used a gene-edited cell line in which all four ATG4 proteases have been knocked out to establish that ATG4A is insufficient to support autophagy and is unable to support GABARAP proteins removal from the membrane. As a result, GABARAP proteins accumulate on membranes other than mature autophagosomes. These results suggest that to support efficient production and consumption of autophagosomes, additional factors are essential including possibly ATG4B itself or one of its proteolytic products in the LC3 family.

Mfa5 from Porphyromonas gingivalis: a von Willebrand factor domain and an intramolecular isopeptide bond in a Gram-negative bacterial fimbrial protein

The Gram-negative bacterium Porphyromonas gingivalis is a secondary colonizer of the oral biofilm and is involved in the onset and progression of periodontitis. Its fimbriae, of type-V, are important for attachment to other microorganisms in the biofilm and for adhesion to host cells. The fimbriae are assembled from five proteins encoded by the mfa1 operon, of which Mfa5 is one of the ancillary tip proteins. Here we report the X-ray structure of the N-terminal half of Mfa5, which reveals a von Willebrand factor domain and two IgG-like domains. One of the IgG-like domains is stabilized by an intramolecular isopeptide bond, which is the first such bond observed in a Gram-negative bacterium. These features make Mfa5 structurally more related to streptococcal adhesins than to the other P. gingivalis Mfa proteins. The structure reported here indicates that horizontal gene transfer has occurred among the bacteria that within the oral biofilm.

Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes

Glutamylation, introduced by TTLL enzymes, is the most abundant modification of brain tubulin. Essential effector proteins read the tubulin glutamylation pattern, and its misregulation causes neurodegeneration. TTLL glutamylases posttranslationally add glutamates to internal glutamates in tubulin C-terminal tails (branch initiation, through an isopeptide bond), and additional glutamates can extend these (elongation). TTLLs are thought to specialize for initiation or elongation, but the mechanistic basis for regioselectivity is unknown. We present cocrystal structures of murine TTLL6 bound to tetrahedral intermediate analogs that delineate key active-site residues that make this an elongase. We show that TTLL4 is exclusively an initiase, and through combined structural and phylogenetic analyses, engineer TTLL6 into a branch-initiating enzyme. TTLL glycylases add glycines posttranslationally to internal glutamates, and we find that the same active-site residues discriminate between initiase and elongase glycylases. These active-site specializations of TTLL glutamylases and glycylases ultimately yield the chemical complexity of cellular microtubules.