To determine the basis for species selectivity, we solved four crystal structures: wild type Mtb proteasome following exposure to GL1 at 2.4 Å resolution and to HT1171 at 2.5 Å resolution, and the open-gate variant (20SOG) alone at 2.5 Å resolution or following exposure to HT1171 at 2.9 Å resolution. N-terminal octa-peptide deletion in the α-subunit of 20SOG did not alter the overall structure of the Mtb proteasome (Fig. S8a
). Furthermore, wild type and open-gate proteasomes underwent the same conformational changes (described below) upon inhibitor treatment (Fig. S8b
). The three structures (, S8, S9
; Table S5
) each confirmed that oxathiazol-2-ones cyclo-carbonylate Thr1. Not only the oxazolidin-2-one ring, but also its protruding methyl group and carbonyl oxygen, were resolved in the electron density (). The oxazolidin-2-one ring is stabilized by an H-bond network involving Ala180, Ser141, Asn24 of the neighboring β-subunit, and a water molecule in the substrate cavity (Fig. S9
). Use of HT1171 and GL1 in the crystallographic studies and HT1171, GL5 and GL3 in the mass spectroscopic studies brought to 4 the number of oxathiazol-2-ones for which the same suicide-substrate inhibition mechanism was confirmed.
Crystal structure of the full-length Mtb 20S proteasome after exposure to HT1171 reveals cyclo-carbonylation of active site Thr1 and conformational changes in the β-subunit
Surprisingly, the substrate-binding pocket of the Mtb proteasome underwent a major conformational change upon cyclo-carbonylation of Thr1 by HT1171 or GL1. Such a change is unprecedented among the dozens of crystal structures of proteasomes in complex with inhibitors11
. An ~8° downward tilt of the H1 helix in the β-subunit moved the N-terminal end of H1 (Ala49-Phe55) downward by as much as 4.2 Å (). The H1 shift brought with it a 3-amino-acid stretch (Ala46-Thr48) of the S4 β-strand, converting this segment into a short loop (S4-H1). As a consequence, another short loop (Met95Gln96Gly97) between H2 and S5 lost stabilizing contacts with the shifted components and became disordered, as illustrated by a black dashed curve in . The S4-H1 loop region comprises the upper surface of the substrate-binding pocket11,15
. Its downward shift constricted the pocket to the point that it could not accommodate a peptide substrate, affording an additional mechanism of inhibition over and above incorporation of the active site hydroxyl into an oxazolidin-2-one.
In the native Mtb β-subunit, the three S4-H1 loop amino acids Ala46, Gly47 and Thr48 are in βstrand configuration and form a β-sheet interaction with Leu101, Ala100 and Leu99, respectively, in the S5 β-strand (). None of these residue pairs is conserved in the human proteasome β5 subunit and only one of them (G47-A96) in the human β1 and β2 subunits. One direct H-bond and three pairs of water-mediated H-bonds stabilized the new position of the S4-H1 loop (): between Glu54 and Trp129 of neighboring β-subunit (3.0 Å); between Thr48, a water, and Asp124 in the S6-S7 loop of neighboring β-subunit (2.8 Å and 3.1 Å, respectively); between Ala50, a water, and the carbonyl O of Trp129 in the short S7 β-strand of the neighboring β-subunit (3.0 Å and 3.1 Å, respectively); and between Ala 49, a water, and Ser20 in the same β-subunit (3.2 Å and 3.1 Å, respectively). This last pair of H-bonds cross-linked the upper and the lower substrate-binding surfaces, sealing the entrance of the substrate pocket. The residue pairs involved in the post-shift H bonds are not well conserved in the human proteasome (). We speculate that the upper substrate-binding surfaces (S4-H1 loops) in the 3 catalytic β-subunits of the human proteasome might have difficulty breaking off from their corresponding β-sheet cores upon initial modification of Thr1 by oxathiazol-2-ones.
Thus, selectivity of oxathiazol-2-ones appears to be imparted in 3 ways: by the presence of a 1,2-aminoalcohol at the active site of the target, which, among enzymes, is likely to be limited to the N-terminal Thr hydrolase family; by the ability of the inhibitor to bind rapidly (before its spontaneous decay) and precisely adjacent to the 1,2-aminoalcohol; and the degree to which the amino group of the 1,2-aminoalcohol has better access to the inhibitor’s carbonyl (now attached to the alcohol) than water has. The protein landscape near the 1,2-aminoalcohol can thus determine species selectivity, both in how it binds the R group on the oxathiazol-2-one and in the conformations it adopts, which may either permit or limit access of water to the intermediate formed during reaction of the oxathiazol-2-one with the active site. Detailed understanding the sequence of steps by which oxathiazol-2-ones cause a major conformational shift in the substrate-binding domain could guide design of the next generation of inhibitors selective for the Mtb proteasome over the human proteasome.