Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins

Abstract

BackgroundThe post-translational modification pathway referred to as pupylation marks proteins for proteasomal degradation in Mycobacterium tuberculosis and other actinobacteria by covalently attaching the small protein Pup (prokaryotic ubiquitin-like protein) to target lysine residues. In contrast to the functionally analogous eukaryotic ubiquitin, Pup is intrinsically disordered in its free form. Its unfolded state allows Pup to adopt different structures upon interaction with different binding partners like the Pup ligase PafA and the proteasomal ATPase Mpa. While the disordered behavior of free Pup has been well characterized, it remained unknown whether Pup adopts a distinct structure when attached to a substrate.ResultsUsing a combination of NMR experiments and biochemical analysis we demonstrate that Pup remains unstructured when ligated to two well-established pupylation substrates targeted for proteasomal degradation in Mycobacterium tuberculosis, malonyl transacylase (FabD) and ketopantoyl hydroxylmethyltransferase (PanB). Isotopically labeled Pup was linked to FabD and PanB by in vitro pupylation to generate homogeneously pupylated substrates suitable for NMR analysis. The single target lysine of PanB was identified by a combination of mass spectroscopy and mutational analysis. Chemical shift comparison between Pup in its free form and ligated to substrate reveals intrinsic disorder of Pup in the conjugate.ConclusionWhen linked to the proteasomal substrates FabD and PanB, Pup is unstructured and retains the ability to interact with its different binding partners. This suggests that it is not the conformation of Pup attached to these two substrates which determines their delivery to the proteasome, but the availability of the degradation complex and the depupylase.Electronic supplementary materialThe online version of this article (doi:10.1186/s12900-017-0072-1) contains supplementary material, which is available to authorized users.

Pupylated proteins are subject to broad proteasomal degradation specificity and differential depupylation

“…MST was used to measure binding of the known genuine Mtb pupylation substrates malonyl transacylase FabD, ketopantoate hydroxymethyltransferase PanB, phosphoenolpyruvate carboxykinase PckA and isocitrate lyase 1 Icl1 to Mpa. To obtain homogeneously pupylated FabD, a triple lysine-to-arginine variant (FabD3KR) was used, removing three secondary pupylation sites and leaving only the main site intact [37]. As the experiments were performed under the exclusion of nucleotide, only coiled-coil interactions between Pup and Mpa, as well as potential unspecific interactions between substrate and Mpa, but not active pore engagement contribute to the binding.…”

“…This demonstrates that the interaction with and subsequent depupylation by Dop is more dependent on the individual substrate than Mpa-mediated binding and translocation into the proteasome. Likely, the interaction of Dop with Pup on a modified target more easily becomes subject to steric clashes at the interface of Dop and substrate, an interpretation also supported by the higher affinity of free Pup compared to Pup-substrate conjugate with Dop [37]. In particular, the placement of the isopeptide bond in the active site might be sterically challenging.…”

“…The regulation contributing to the fate of a pupylated protein could occur at multiple levels, including the interplay of pupylation and depupylation, but also the recruitment and degradation of pupylated substrates by the proteasome complex itself [5, 26, 35, 36]. Using a combination of NMR experiments and biochemical analysis we have recently shown that the intrinsically disordered Pup remains unstructured when ligated to two well-established pupylation substrates, retaining the ability to interact with its different interaction partners and ruling out the conformation of Pup on the substrate as a decisive element for substrate fate [37].…”

T cell receptor sequence clustering and antigen specificity

“…Despite the difficulties of modelling flexible loops, such as the CDR regions of the TCR, several tools have been explored, and the field is an active area of research. Models predicting TCR-pMHC binding based on their structure have already been investigated [15] , [17] , [18] , [19] , [20] .…”

Prokaryotic Ubiquitin-Like Protein and Its Ligase/Deligase Enyzmes

“…Post-translational modification, specifically phosphorylation has been suggested as one possibility [66]. Alternatively, it is also possible that an additional, conformational journey with a range of disorder-to-order transitions [67]. As the next paragraph will show, its interaction with the modification enzymes requires yet a different conformation.…”