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, 15 (7), 1701-9

Structural Analysis of an "Open" Form of PBP1B From Streptococcus Pneumoniae


Structural Analysis of an "Open" Form of PBP1B From Streptococcus Pneumoniae

Andrew L Lovering et al. Protein Sci.


The class A PBP1b from Streptococcus pneumoniae is responsible for glycosyltransferase and transpeptidase (TP) reactions, forming the peptidoglycan of the bacterial cell wall. The enzyme has been produced in a stable, soluble form and undergoes time-dependent proteolysis to leave an intact TP domain. Crystals of this TP domain were obtained, diffracting to 2.2 A resolution, and the structure was solved by using molecular replacement. Analysis of the structure revealed an "open" active site, with important conformational differences to the previously determined "closed" apoenzyme. The active-site nucleophile, Ser460, is in an orientation that allows for acylation by beta-lactams. Consistent with the productive conformation of the conserved active-site catalytic residues, adjacent loops show only minor deviation from those of known acyl-enzyme structures. These findings are discussed in the context of enzyme functionality and the possible conformational sampling of PBP1b between active and inactive states.


Figure 1.
Figure 1.
SDS-PAGE of truncated PBP1b protein samples. A 10% (w/v) acrylamide separating gel was used, with subsequent steps following standard procedure. (Lane 1) High-range molecular markers (Bio-Rad; molecular weight given in kilodaltons). (Lane 2) “Freshly prepared” truncated PBP1b protein stock. (Lane 3) Moenomycin-aged truncated PBP1b protein stocks. The aging of protein samples was carried out in an attempt to mimic any proteolysis occurring in the crystallization drop. N-terminal protein sequencing results were used to characterize the protein bands: Lane 2, upper band MD85KVRV, lower band L184IKQQV; lane 3, upper band Q323DFLPS, lower band K139AIIAT. The desired soluble truncation product of our PBP1b construct was present in freshly prepared protein samples, starting at the native sequence residue D85 (post-transmembrane helix). This sample also contains a contaminating band starting at L184, resulting from proteolysis of the GT domain. After protein aging, the larger, two-domain constructs are pared down to the linker region and TP domain (Q323 onward). The smaller, TP-associated hairpin from the GT domain (to the smallest possible extent, residues 105–119) is not visible on this gel. If the degradation observed in aged samples is comparable to that in the crystallization drop, residues 323–336 must be disordered in the electron density map. It is also possible that further proteolysis has occurred precrystallization at the protease-sensitive R336 position (Macheboeuf et al. 2005).
Figure 2.
Figure 2.
Active-site loop movement in PBP1b. (A) Electron density for active-site loop of the truncated PBP1b apoenzyme. Figure colored according to atom type (yellow indicates C; red, O; blue, N; orange, S). Map is from a 2Fo − Fc calculation omitting residues 652–662, contoured around these residues at 1σ level to 2.2 Å resolution. (B) Comparison of the Cα-traces for open, closed, and acylated PBP1b TP domains. Structures were aligned using the auto-fit procedure of SwissPDBViewer (Guex and Peitsch 1997). Open structure is of the apoenzyme reported in this study (PDB 2FFF, yellow). Closed apoenzyme structure (PDB 2BG1, blue) and nitrocefin acyl-enzyme (PDB 2BG3, green) are from results reported by Macheboeuf et al. (2005). The side chain of the active site S460 nucleophile from the open structure is shown in stick form (C atoms, gray; Oγ atom, red). Major differences between three active-site loops can be observed between the open/acyl and the closed forms (within red ellipse; residues 414–421, 653–660, and 677–687). Differences can also be observed at the mobile N-terminal domain (within magenta ellipse), especially in helices Ha and Hb (residues 337–358 and 369–384, respectively).
Figure 3.
Figure 3.
Detail of PBP1b active-site differences, with selected side-chain residues shown in stick form. Figure colored according to atom type (yellow indicates open PBP1b structure C; purple, closed PBP1b C; green, acyl-enzyme PBP1b C; gray, PBP1a C; , red, O; blue, N; orange, S). (A) Comparison of the open (PDB 2FFF [this study]) and closed (PDB 2BG1 [Macheboeuf et al. 2005]) apoenzyme structures. Labeling with a prime denotes residues from the closed structure. The figure shows the large shift in the polypeptide backbone of loop β3–β4 (AA 653–660) and shifts in the position of the important active-site residues T654, T655, N656, and Q657. These changes between the two apoenzyme structures result in the occlusion of the active site in the closed form, largely due to N656 blocking entry of substrate. The S460 nucleophile is unavailable for reaction in the closed form, making close contacts to the backbone region of β3 (at the oxyanion hole formed between the N atoms of S460 and T654). With the shift of the active-site loops, the antiparallel nature of β3 and β4 is disturbed in the closed form, resulting in the premature termination of strand β3. The conformations of residues R686 and R687 are also shown, differing from the glutamine side chains of the closed form construct. These mutations occur in a protease-sensitive area of the molecule and may be responsible in part for observed kinetic differences to the wild type enzyme (Macheboeuf et al. 2005). (B) Comparison of the open (this study) and nitrocefin acyl-enzyme (PDB 2BG3 [Macheboeuf et al. 2005]) structures. Nitrocefin moiety of acyl-enzyme shown as partly transparent space-fill model. The backbone atoms are in closer agreement with our structure than those of the closed apoenzyme (A), and the antiparallel nature of β3 and β4 is restored. Most of the active-site side chains are in a similar conformation between the two structures, with T654, T655, N656, and Q657 only needing minor rotametric alterations to achieve complementarity. The open form shows very little steric clash with the placement of the nitrocefin adduct, with only R686 needing a significant movement to accommodate the bulky R2 nitro group. The side chain of the active-site S460 nucleophile is also in a similar conformation between the open and the acyl-enzyme structures. Such general agreement between the two forms validates the assumption that the apoenzyme presented in this study is in a more productive conformation than that previously observed (Macheboeuf et al. 2005). (C) Comparison of the open PBP1b apoenzyme (this study) and the PBP1a (PDB 2C6W [Contreras-Martel et al. 2006]) structures. The similarity in position of the nucleophile (S460 PBP1b, S370 PBP1a) confirms the differences observed between this apoenzyme and that from previous studies (B). Residues lining the active site (T654/T655/N656 for PBP1b; T560/S561/N562 for PBP1a) also show good agreement, despite the insertion of a helix in loop b3/b4, and the change in topology of some of the active-site loops.

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