The pH-Dependent Trigger in Diphtheria Toxin T Domain Comes with a Safety Latch.

Article The pH-Dependent Trigger in Diphtheria Toxin T Domain Comes with a Safety Latch Mykola V. Rodnin,1 Jing Li,2 Michael L. Gross,2 and Alexey S. Ladokhin1,* 1 Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas; and 2Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri ABSTRACT Protein-side-chain protonation, coupled to conformational rearrangements, is one way of regulating physiological function caused by changes in protein environment. Specifically, protonation of histidine residues has been implicated in pH-dependent conformational switching in several systems, including the diphtheria toxin translocation (T) domain, which is responsible for the toxin’s cellular entry via the endosomal pathway. Our previous studies a) identified protonation of H257 as a major component of the T domain’s conformational switch and b) suggested the possibility of a neighboring H223 acting as a modulator, affecting the protonation of H257 and preventing premature conformational changes outside the endosome. To verify this ‘‘safety-latch’’ hypothesis, we report here the pH-dependent folding and membrane interactions of the T domain of the wild-type and that of the H223Q mutant, which lacks the latch. Thermal unfolding of the T domain, measured by circular dichroism, revealed that the reduction in the transition temperature for helical unfolding for an H223Q mutant starts at less acidic conditions (pH <7.5) relative to the wild-type protein (pH <6.5). Hydrogen-deuterium-exchange mass spectrometry demonstrates that the H223Q replacement results in a loss of stability of the amphipathic helices TH1–3 and the hydrophobic core helix TH8 at pH 6.5. That this destabilization occurs in solution correlates well with the pH-range shift for the onset of the membrane permeabilization and translocation activity of the T domain, confirming our initial hypothesis that H223 protonation guards against early refolding. Taken together, these results demonstrate that histidine protonation can fine-tune pH-dependent switching in physiologically relevant systems. INTRODUCTION Conformational signaling, broadly defined as structural switching in response to external stimuli, is involved in all aspects of cellular functioning. Changes in proton concentration are among the most prominent physicochemical signals capable of triggering functionally relevant structural rearrangements. pH-dependent transitions play important roles in the cellular entry of bacterial toxins (1–5), colicins (6), and viruses (7,8), in the function of pH-sensors (9,10), in the signal cascades of G-protein (11), and possibly in apoptotic regulation (12–16). Protonation-dependent transitions are also involved in cancer pathogenesis, because ‘‘the dysregulation of pH by cancerous cells is able to create a unique milieu that is in favor of progression, invasion, and metastasis as well as chemo-/immunoresistance of solid tumors’’ (17). On the other hand, novel approaches for the delivery of anticancer therapy and tumor imaging are using Submitted March 24, 2016, and accepted for publication September 19, 2016. *Correspondence: Editor: Andreas Engel. http://dx.doi.org/10.1016/j.bpj.2016.09.030 Ó 2016 Biophysical Society. 1946 Biophysical Journal 111, 1946–1953, November 1, 2016 pH-dependent transitions for targeting diseased acidic tissues (18). Although the exact mechanism for coupling protonation and conformational changes remains largely unknown, protonation of histidine residues does cause conformational switching in several systems (7,8,19,20), consistent with order-disorder transitions in intrinsically disordered proteins and their fragments (21–23). Specifically, histidines do play an important role in the functioning of the diphtheria toxin translocation domain (T domain) (24–28), making it an attractive model for studies of mechanisms of conformational switching by protonation (29). Diphtheria toxin enters the cell via the endosomal pathway, which is shared by many other toxins, including botulinum, tetanus, and anthrax (1–5). A crucial step in cell infection is the translocation of the catalytic domain from the endosome into the cytosol, accomplished by the translocation domain. Diphtheria toxin T domain is a small, 178-residue protein fragment that forms a separate, tightly folded domain at neutral pH but changes its conformation under the acidic conditions inside the endosome. It then inserts into the lipid bilayer and translocates its own N-terminus and the attached catalytic domain across the membrane pH-Dependent Conformational Switching (30,31). Although the crystallographic structure of the soluble toxin is known (32), as are the interactions of the T domain with lipid bilayers (24,33–43), we report here a significantly improved understanding of the molecular mechanism of its action. Previously, we demonstrated that the protonation of H257 is a principal element of the pH-dependent trigger, responsible for destabilizing the solution fold of the T domain in preparation for its membrane insertion (25). Our recent computational study confirmed this conclusion by estimating the free-energy penalty for protonating all six histidine residues in the T domain by thermodynamic integration along the molecular dynamics (MD) simulation trajectory (28). Protonation of H257 has the highest free-energy penalty and, hence, the most destabilizing effect on the native structure. In contrast, protonation of the neighboring H223 is favorable, suggesting that this residue is easily protonated without causing any structural rearrangements. The protonation of H223, however, has an effect on the freeenergy penalty for protonation of H257, increasing it by ~3 kcal/mole. Similar conclusions follow from an independent calculation of pKa distributions based on the continuum electrostatic model, resulting in the shift of the distribution of pKas in an MD-generated ensemble toward acidic pH by ~1.5 units (equivalent to 2 kcal/mole in terms of free energy) (28). Safety-latch hypothesis of conformational switching in diphtheria toxin T domain Acidification of the endosomal environment triggers a series of conformational changes in the T domain, ultimately resulting in its refolding and bilayer insertion. The first stage occurs in solution and results in a pH-dependent conversion of the membrane-incompetent W-state into a protonated membrane-competent Wþ-state. The latter state is prone to aggregation, which significantly complicates its structural characterization and hampers applications of high-resolution methods. Recently, we elucidated conformational changes occurring during the W-to-Wþ transition using MD simulations and fluorescence and circular dichroism spectroscopy (CD), and used various computational approaches to estimate the pKas for the T domain’s six histidine residues (28). The resulting distributions of pKa probabilities for both W- and Wþ-states (...truncated)