Plant and animal pathogenic bacteria can suppress host immunity by injecting type III secreted effector (T3SE) proteins into host cells. However, T3SEs can also elicit host immunity if the host has evolved a means to recognize the presence or activity of specific T3SEs. The diverse YopJ/HopZ/AvrRxv T3SE superfamily, which is found in both animal and plant pathogens, provides examples of T3SEs playing this dual role. The T3SE HopZ1a is an acetyltransferase carried by the phytopathogen Pseudomonas syringae that elicits effector-triggered immunity (ETI) when recognized in Arabidopsis thaliana by the nucleotide-binding leucine-rich repeat (NB-LRR) protein ZAR1. However, recognition of HopZ1a does not require any known ETI-related genes. Using a forward genetics approach, we identify a unique ETI-associated gene that is essential for ZAR1-mediated immunity. The hopZ-ETI-deficient1 (zed1) mutant is specifically impaired in the recognition of HopZ1a, but not the recognition of other unrelated T3SEs or in pattern recognition receptor (PRR)-triggered immunity. ZED1 directly interacts with both HopZ1a and ZAR1 and is acetylated on threonines 125 and 177 by HopZ1a. ZED1 is a nonfunctional kinase that forms part of small genomic cluster of kinases in Arabidopsis. We hypothesize that ZED1 acts as a decoy to lure HopZ1a to the ZAR1-resistance complex, resulting in ETI activation.ZED1-related kinase | ZRK | hypersensitive response

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Charge reduction in the gas phase provides a direct means of manipulating protein charge state, and when coupled to ion mobility mass spectrometry (IM-MS), it is possible to monitor the effect of charge on protein conformation in the absence of solution. Use of the electron transfer reagent 1,3-dicyanobenzene, coupled with IM-MS, allows us to monitor the effect of charge reduction on the conformation of two proteins deliberately chosen from opposite sides of the order to disorder continuum: bovine pancreatic trypsin inhibitor (BPTI) and beta casein. The ordered BPTI presents compact conformers for each of three charge states accompanied by narrow collision cross-section distributions (TWCCSDN2→He). Upon reduction of BPTI, irrespective of precursor charge state, the TWCCSN2→He decreases to a similar distribution as found for the nESI generated ion of identical charge. The behavior of beta casein upon charge reduction is more complex. It presents over a wide charge state range (9–28), and intermediate charge states (13–18) have broad TWCCSDN2→He with multiple conformations, where both compaction and rearrangement are seen. Further, we see that the TWCCSDN2→He of the latter charge states are even affected by the presence of radical anions. Overall, we conclude that the flexible nature of some proteins result in broad conformational distributions comprised of many families, even for single charge states, and the barrier between different states can be easily overcome by an alteration of the net charge. Graphical Abstractᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-017-1692-1) contains supplementary material, which is available to authorized users.

The gas phase is an idealized laboratory for the study of protein structure, from which it is possible to examine stable and transient forms of mass-selected ions in the absence of bulk solvent. With ion mobility–mass spectrometry (IM-MS) apparatus built to operate at both cryogenic and elevated temperatures, we have examined conformational transitions that occur to the monomeric proteins: ubiquitin, lysozyme, and α-synuclein as a function of temperature and in source activation. We rationalize the experimental observations with a temperature-dependent framework model and comparison to known conformers. Data from ubiquitin show unfolding transitions that proceed through diverse and highly elongated intermediate states, which converge to more compact structures. These findings contrast with data obtained from lysozyme—a protein where (un)-folding plasticity is restricted by four disulfide linkages, although this is alleviated in its reduced form. For structured proteins, collision activation of the protein ions in-source enables subsequent “freezing” or thermal annealing of unfolding intermediates, whereas disordered proteins restructure substantially at 250 K even without activation, indicating that cold denaturation can occur without solvent. These data are presented in the context of a toy model framework that describes the relative occupancy of the available conformational space.

<p>Using Ubiquitin as an exemplar protein we examine the effect of net charge reduction post electrospray ionisation by exposure to the electron transfer reagent, 1,3-dicyanobenzene. We monitor the change in gas phase conformation of both precursor and products with ion mobility mass spectrometry (IM-MS). Dramatic conformational rearrangement is seen for low charge state ions upon exposure to the electron transfer reagent. Ions with low charge states sprayed from both native-like and denaturing solvent conditions undergo structural transitions to conformers with cross sections in the range measured for the native structure (^TWCCS_N2_®_He, 950-1000 Ų). Thus, we infer that some memory of the solution phase structure is retained in the gas phase. Intermediate structures are seen in the reduction of the [M+6H]⁶⁺ ion sprayed from both native-like and denaturing solvents. Further, the reduction pathway of this ion shows compaction to structures with a ^TWCCS_N2_®_He centred at 1069 Ų (5+) and 949 Ų (4+) for ions originating from native-like and denaturing solvents respectively. We propose that charge reduction sites for radical anion localisation (to effect electron transfer) are not easily accessible in the case of ubiquitin molecules originating from native-like solution conditions. This highlights the importance of salt bridge interactions in maintaining the structural integrity of a protein in the gas phase. Most interestingly, two distinct conformer populations are seen for the 6+ charge-reduced product originating from the 7+ and, the 6+ exposed to radical anions (post ESI); we infer that these populations are intermediate in the refolding of ubiquitin in the gas phase, sometimes transient. Overall, we are able to monitor the refolding pathway of ubiquitin in the gas phase as its charge is reduced and show that charge-charge interactions play a significant role in the gas phase conformation adopted; whereby specific conformations are formed. </p>