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Legionella pneumophila establishes a replication vacuole by translocating hundreds of protein effectors through a type IV secretion system (T4SS). Among these translocated effectors are members of the Sde family, which catalyze phosphoribosyl-linked ubiquitination (pR-Ub) of host targets. Previous work has posited that Sde proteins solely target serine (Ser) residues within acceptor protein substrates. We show here that SdeC-mediated pR-Ub modification results from a stepwise reaction that also modifies tyrosine (Tyr) residues. Unexpectedly, the presence of an HA tag on Ub resulted in poly-pR-ubiquitination, consistent with the HA tag acting as an acceptor target. Interrogation of phosphoribosyl-linked HA-Ub revealed that Tyr4 was the preferred targeted residue, based on LC-MS/MS analysis of the crosslinked product. Further analysis using synthetic HA variants revealed promiscuous modification of Tyr, as crosslinking was prevented only by constructing a triple mutant in which all three Tyr within the HA sequence were substituted with Phe. Although previous work has indicated that Ser is the sole acceptor residue, we found no evidence of Ser preference over Tyr using Tyr → Ser replacement mutants. This work demonstrates that pR-ubiquitination by the Sde family is not limited to Ser-modification as previously proposed, and broadens the potential sites targeted by this family., During infection, Legionella pneumophila translocates hundreds of effectors into host cells. Among these, the Sde family effector SdeC catalyzes atypical ubiquitination of host targets at tyrosine, not only serine, residues.

Glycation is a non-enzymatic post-translational modification (PTM) that remains poorly understood, largely because it is unknown how it occurs selectively. Using mass spectrometry, it was possible to evaluate total glycation levels, identify distinct glycated products, assign unique glycation sites, and correlate these data with chemical and structural features for a panel of proteins glycated in vitro. It was determined that the extent of glycation does not correlate with pKa or surface exposure at reactive sites. Rather, the data reveal that primary sequence dictates the overall likelihood that a site will become glycated, while surrounding structure further sculpts the glycation outcome. Clustered acidic residues were found to prevent glycation, whereas a combination of tyrosine and polar residues appear to promote glycation. This work contributes important new knowledge about the molecular features that govern selective glycation.

Bacterial phosphothreonine lyases, or phospholyases, catalyze a unique post-translational modification that introduces dehydrobutyrine (Dhb) or dehydroalanine (Dha) in place of phosphothreonine or phosphoserine residues, respectively. We report the use of a phospha-Michael reaction to label proteins and peptides modified with Dha or Dhb. We demonstrate that a nucleophilic phosphine probe is able to modify Dhb-containing proteins and peptides that were recalcitrant to reaction with thiol or amine nucleophiles under mild aqueous conditions. Furthermore, we used this reaction to detect multiple Dhb-modified proteins in mammalian cell lysates, including histone H3, a previously unknown target of phospholyases. This method should prove useful for identifying new phospholyase targets, profiling the biomarkers of bacterial infection, and developing enzyme-mediated strategies for bioorthogonal labeling in living cells.

Phosphothreonine lyases are bacterial effector proteins secreted into host cells to facilitate the infection process. This enzyme family catalyzes an irreversible elimination reaction that converts phosphothreonine or phosphoserine to dehydrobutyrine or dehydroalanine, respectively. Herein, we report a study of substrate selectivity for each of the four known phosphothreonine lyases. This was accomplished using a combination of mass spectrometry and enzyme kinetics assays for a series of phosphorylated peptides derived from the mitogen-activated protein kinase (MAPK) activation loop. These studies provide the first experimental evidence that VirA, a putative phosphothreonine lyase identified through homology, is indeed capable of catalyzing phosphate elimination. These studies further demonstrate that OspF is the most promiscuous phosphothreonine lyase, whereas SpvC is the most specific for the MAPK activation loop. Our studies reveal that phospholyases are dramatically more efficient at catalyzing elimination from phosphothreonine than from phosphoserine. Together, our data suggest that each enzyme likely has preferred substrates, either within the MAPK family or beyond. Fully understanding the extent of selectivity is key to understanding the impact of phosphothreonine lyases during bacterial infection and to exploiting their unique chemistry for a range of applications.

Intracellular pathogens manipulate host organelles to support replication within cells. For Legionella pneumophila, the bacterium translocates proteins that establish an endoplasmic reticulum (ER)-associated replication compartment. We show here that the bacterial Sde proteins target host reticulon 4 (Rtn4) to control tubular ER dynamics, resulting in tubule rearrangements as well as alterations in Rtn4 associated with the replication compartment. These rearrangements are triggered via Sde-promoted ubiquitin transfer to Rtn4, occurring almost immediately after bacterial uptake. Ubiquitin transfer requires two sequential enzymatic activities from a single Sde polypeptide: an ADP-ribosyltransferase and a nucleotidase/phosphohydrolase. The ADP-ribosylated moiety of ubiquitin is a substrate for the nucleotidase/phosphohydrolase, resulting in either transfer of ubiquitin to Rtn4, or phosphoribosylation of ubiquitin in the absence of a ubiquitination target. Therefore, a single bacterial protein drives a multistep biochemical pathway to control ubiquitination and tubular ER function independently of the host ubiquitin machinery.

Aberrant activation of the epidermal growth factor receptor (EGFR), a prototypic receptor tyrosine kinase, is critical to the biology of many common cancers. The molecular events that define how EGFR transmits an extracellular ligand binding event through the membrane are not understood. Here we use a chemical tool, bipartite tetracysteine display, to report on ligand-specific conformational changes that link ligand binding and kinase activation for full-length EGFR on the mammalian cell surface. We discover that EGF binding is communicated to the cytosol through formation of an antiparallel coiled coil within the intracellular juxtamembrane (JM) domain. This conformational transition is functionally coupled to receptor activation by EGF. In contrast, TGFα binding is communicated to the cytosol through formation of a discrete, alternative helical interface. These findings suggest that the JM region can differentially decode extracellular signals and transmit them to the cell interior. Our results provide new insight into how EGFR communicates ligand-specific information across the membrane.

Chemical reactions that facilitate the attachment of synthetic groups to proteins are useful tools for the field of chemical biology and enable the incorporation of proteins into new materials. We have previously reported a pyridoxal 5'-phosphate (PLP)-mediated reaction that site-specifically oxidizes the N-terminal amine of a protein to afford a ketone. This unique functional group can then be used to attach a reagent of choice through oxime formation. Since its initial report, we have found that the N-terminal sequence of the protein can significantly influence the overall success of this strategy. To obtain short sequences that lead to optimal conversion levels, an efficient method for the evaluation of all possible N-terminal amino acid combinations was needed. This was achieved by developing a generalizable combinatorial peptide library screening platform suitable for the identification of sequences that display high levels of reactivity toward a desired bioconjugation reaction. In the context of N-terminal transamination, a highly reactive alanine-lysine motif emerged, which was confirmed to promote the modification of peptide substrates with PLP. This sequence was also tested on two protein substrates, leading to substantial increases in reactivity relative to their wild-type termini. This readily encodable tripeptide thus appears to provide a significant improvement in the reliability with which the PLP-mediated bioconjugation reaction can be used. This study also provides an important first example of how synthetic peptide libraries can accelerate the discovery and optimization of protein bioconjugation strategies.

For a range of protein substrates, N-terminal transamination offers a convenient way to install a reactive ketone or aldehyde functional group at a single location. We report herein the effects of the identity of N-terminal residues on the product distribution generated upon reaction with pyridoxal 5'-phosphate (PLP). This study was accomplished through the combination of solid-phase peptide synthesis with detailed liquid chromatography-mass spectrometry analysis. Many N-terminal amino acids provided high yields of the desired transaminated products, but some residues (His, Trp, Lys, and Pro) generated adducts with PLP itself. N-terminal Cys and Ser residues were observed to undergo beta-elimination in addition to transamination, and the transamination product of N-terminal Gln was resistant to subsequent oxime formation attempts. The information generated through the screening of peptide substrates was successfully applied to a protein target, changing an initially unreactive terminus into one that could be modified in high (70%) yield. Thus, these studies have increased our predictive power for the reaction, both in terms of improving conversion and suppressing reaction byproducts. An initial set of guidelines that may be used to increase the applicability of this reaction to specific proteins of interest is provided.

SummaryBinding of transforming growth factor α (TGF-α) to the epidermal growth factor receptor (EGFR) extracellular domain is encoded through the formation of a unique antiparallel coiled coil within the juxtamembrane segment. This new coiled coil is an “inside-out” version of the coiled coil formed in the presence of epidermal growth factor (EGF). A third, intermediary coiled-coil interface is formed in the juxtamembrane region when EGFR is stimulated with betacellulin. The seven growth factors that activate EGFR in mammalian systems (EGF, TGF-α, epigen, epiregulin, betacellulin, heparin-binding EGF, and amphiregulin) fall into distinct categories in which the structure of the coiled coil induced within the juxtamembrane region correlates with cell state. The observation that coiled-coil state tracks with the downstream signaling profiles for each ligand provides evidence for growth factor functional selectivity by EGFR. Encoding growth factor identity in alternative coiled-coil rotamers provides a simple and elegant method for communicating chemical information across the plasma membrane.

A convenient new method is described for the introduction of ketone groups at the N-termini of antibodies. The reaction occurs in the presence of pyridoxal-5′-phosphate under conditions mild enough to maintain antigen binding function, as confirmed by enzyme-linked immunosorbent assay. Further derivatization of these functional sites was accomplished through oxime formation, yielding well-defined antibody conjugates for a wide range of applications. The ability of the modified antibodies to bind their targets was confirmed via immunodot blot analysis. The generality of this method has been demonstrated on a number of monoclonal and polyclonal antibodies, all with different binding specificities.