Crystal Structure of the 47-kDa Lipoprotein of Treponema pallidum Reveals a Novel Penicillin-binding Protein

Abstract

Syphilis is a complex sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum. T. pallidum has remained exquisitely sensitive to penicillin, but the mode of action and lethal targets for ␤-lactams are still unknown. We previously identified the T. pallidum 47-kDa lipoprotein (Tp47) as a penicillin-binding protein (PBP). Tp47 contains three hypothetical consensus motifs (SVTK, TEN, and KTG) that typically form the active center of other PBPs. Yet, in this study, mutations of key amino acids within these motifs failed to abolish the penicillin binding activity of Tp47. The crystal structure of Tp47 at a resolution of 1.95 Å revealed a fold different from any other known PBP; Tp47 is predominantly ␤-sheet, in contrast to the ␣/␤-fold common to other PBPs. It comprises four distinct domains: two complex ␤-sheet-containing N-terminal domains and two C-terminal domains that adopt immunoglobulin-like folds. The three hypothetical PBP signature motifs do not come together to form a typical PBP active site. Furthermore, Tp47 is unusual in that it displays ␤-lactamase activity (k cat for penicillin ‫؍‬ 271 ؎ 6 s ؊1 ), a feature that hindered attempts to identify the active site in Tp47 by co-crystallization and mass spectrometric techniques. Taken together, Tp47 does not fit the classical structural and mechanistic paradigms for PBPs, and thus Tp47 appears to represent a new class of PBP.

TprC/D (Tp0117/131), a Trimeric, Pore-Forming Rare Outer Membrane Protein of Treponema pallidum, Has a Bipartite Domain Structure

“…In our recent study of TP0326/BamA (19), we determined the mean copy number of tprC transcript to be 212 Ϯ 35 per 10,000 copies of flaA. Although more than 3-fold greater than levels of tp0326 transcript (61 Ϯ 16), this value is markedly lower than the more than 2,500 flaA-normalized copies of tp0547 message, which encodes the abundant lipoprotein carboxypeptidase Tp47 (17,19). Expression of TprC on a per-cell basis was here determined by quantitative immunoblot analysis (Fig.…”

The Tp38 (TpMglB-2) Lipoprotein Binds Glucose in a Manner Consistent with Receptor Function in Treponema pallidum

“…3). Changes in the intrinsic fluorescence of recombinant TroA, a periplasmic ABC metal-binding protein of T. pallidum (8,19), or Tp47, a T. pallidum periplasmic penicillin-binding protein (9,46), in the presence of D-glucose were below 2% (not shown). In comparison with all of these baseline (negative control) values, the intrinsic fluorescence of Tp38 changed markedly only in the presence of D-glucose or D-galactose (Fig.…”

“…Recombinant TroA was purified from E. coli as previously described (8). Recombinant Tp47 was isolated according to previously published methods (9).…”

Global Challenge of Antibiotic-Resistant Treponema pallidum

“…Analysis of the T. pallidum genome sequence predicts three putative PBPs but no typical ␤-lactamases (17). However, Cha et al (8) showed that Tp47, an abundant, membrane-bound lipoprotein that was initially identified and characterized by Norgard and colleagues (12,82), binds penicillin and has high ␤-lactamase activity that is subject to strong product inhibition. Furthermore, Cha et al (8) hypothesized that if a mutant variant of Tp47 emerges that overcomes the product inhibition of its ␤-lactamase activity, this would confer novel, bona fide resistance to penicillin.…”