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Repeats-in-Toxin (RTX) proteins of Gram-negative bacteria are excreted through the type I secretion system (T1SS) that recognizes non-cleavable C-terminal secretion signals. These are preceded by arrays of glycine and aspartate-rich nonapeptide repeats grouped by four to eight β strands into blocks that fold into calcium-binding parallel β-roll structures. The β-rolls are interspersed by linkers of variable length and sequence and the organization of multiple RTX repeat blocks within large RTX domains remains unknown. Here we examined the structure and function of the RTX domain of Bordetella pertussis adenylate cyclase toxin (CyaA) that is composed of five β-roll RTX blocks. We show that the non-folded RTX repeats maintain the stability of the CyaA polypeptide in the Ca2+-depleted bacterial cytosol and thereby enable its efficient translocation through the T1SS apparatus. The efficacy of secretion of truncated CyaA constructs was dictated by the number of retained RTX repeat blocks and depended on the presence of extracellular Ca2+ ions. We further describe the crystal structure of the RTX blocks IV-V of CyaA (CyaA1372-1681) that consists of a contiguous assembly of two β-rolls that differs substantially from the arrangement of the RTX blocks observed in RTX lipases or other RTX proteins. These results provide a novel structural insight into the architecture of the RTX domains of large RTX proteins and support the "push-ratchet" mechanism of the T1SS-mediated secretion of very large RTX proteins.

Bioorganic & medicinal chemistry 27(2), 255 - 264 (2019). doi:10.1016/j.bmc.2018.11.022, A series of carbamate-based inhibitors of glutamate carboxypeptidase II (GCPII) were designed and synthesizedusing ZJ-43, N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-L-glutamic acid, as a molecular template inorder to better understand the impact of replacing one of the two nitrogen atoms in the urea-based GCPIIinhibitor with an oxygen atom. Compound 7 containing a C-terminal 2-oxypentanedioic acid was more potentthan compound 5 containing a C-terminal glutamic acid (2-aminopentanedioic acid) despite GCPII’s preferencefor peptides containing an N-terminal glutamate as substrates. Subsequent crystallographic analysis revealedthat ZJ-43 and its two carbamate analogs 5 and 7 with the same (S,S)-stereochemical configuration adopt anearly identical binding mode while (R,S)-carbamate analog 8 containing a D-leucine forms a less extensivehydrogen bonding network. QM and QM/MM calculations have identified no specific interactions in the GCPIIactive site that would distinguish ZJ-43 from compounds 5 and 7 and attributed the higher potency of ZJ-43 andcompound 7 to the free energy changes associated with the transfer of the ligand from bulk solvent to the proteinactive site as a result of the lower ligand strain energy and solvation/desolvation energy. Our findings underscorea broader range of factors that need to be taken into account in predicting ligand-protein binding affinity.These insights should be of particular importance in future efforts to design and develop GCPII inhibitors foroptimal inhibitory potency., Published by Elsevier, Amsterdam [u.a.]

Molecular cell 62(1), 47 - 62 (2016). doi:10.1016/j.molcel.2016.03.018, Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca$^{2+}$-depleted cytosol of Gram-negative bacteria directly into the Ca$^{2+}$-rich external milieu, passing through the “channel-tunnel” ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca$^{2+}$-dependent folding of the carboxy-proximal RTX repeat blocks into β-rolls. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial “push-ratchet” mechanism. Successive Ca$^{2+}$-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens., Published by Elsevier, Cambridge, Mass.