Elucidation of the structure of the membrane anchor of penicillin-binding protein 5 of Escherichia coli

Abstract

Penicillin-binding protein 5 (PBP 5) of Escherichia coli is a membrane-bound cell wall dd-carboxypeptidase, localized in the outer leaflet of the cytosolic membrane of this Gram-negative bacterium. Not only is it the most abundant PBP of E. coli, but it is as well a target for penicillins and is the most studied of the PBP enzymes. PBP 5, as a representative peripheral membrane protein, is anchored to the cytoplasmic membrane by the 21 amino acids of its C-terminus. Although the importance of this terminus as a membrane anchor is well recognized, the structure of this anchor was previously unknown. Using natural isotope abundance NMR, the structure of the PBP 5 anchor peptide within a micelle was determined. The structure conforms to a helix-bend-helix-turn-helix motif and reveals that the anchor enters the membrane so as to form an amphiphilic structure within the interface of the hydrophilic/hydrophobic boundary regions near the lipid head groups. The bend and the turn within the motif allow the C-terminus to exit from the same side of the membrane that is penetrated. The PBP anchor sequences represent extraordinary diversity, encompassing both N-terminal and C-terminal anchoring domains. This study establishes a surface adherence mechanism for the PBP 5 C-terminus anchor peptide, as the structural basis for further study toward understanding the role of these domains in selecting membrane environments and in the assembly of the multienzyme hyperstructures of bacterial cell wall biosynthesis.

Figure 1

(A) The ¹H-¹⁵N HSQC spectrum of DPC micelle-bound PBP 5 anchor for the ¹H δ 10.5–7.0 ppm and ¹⁵N δ 105–130 regions. The cross-peaks are labeled by the amino acid residue. The inset is an expansion of the ¹H δ 8.5–8.0 ppm and ¹⁵N δ 117–121 ppm region. (B) Chemical shift index for the α-protons of the PBP 5 anchor. Values between –0.10 and 0.10 are considered zero. Negative values indicate helical structure and positive values indicate beta structure. (C) Chemical shift index for ¹³C α-carbons of the PBP 5 anchor. Values between –0.7 and 0.7 are considered zero. Negative values indicate beta sheet structure and positive values indicate helical structure.

Figure 2

NOE-connectivity table and stereo view of the peptide backbone. (A) Summary of NOE connectivities from NOESY spectrum (mixing time 200 ms) of PBP 5 anchor in DPC micelle (1:55) at pH 7.4 and 25.0 °C. The thickness of the band (strong, medium and weak) corresponds to the intensity of the NOE interaction between residues. The first four rows indicate interactions between adjacent amino acids. The remaining rows show interactions between the residues at each end of the bar. (B) Stereo view of the 20 overlaid backbone structures of the PBP 5 anchor from the Cyana calculation. The N-terminus of the peptide is on the top. The first three N-terminal residues display several conformations, while two distinct conformations are seen for the Gly21 C-terminus residue of the peptide.

Figure 3

Structure of the E. coli PBP 5 anchor peptide. (A) The ribbon structure of the PBP 5 anchor displaying its secondary structure. The surface domain of the protein connects to the N-terminus (right side) of the peptide. The α-helical portions of the peptide are colored red and yellow and the non-α-helical portions are displayed as wires in gray. (B) Stereo view of the peptide (backbone as a wire in green) with side chains displayed as capped sticks (C in gray; O in red; N in blue; S in yellow). Hydrogens are removed and the residues are labeled for clarity. (C) Stereo view of the computational model of the peptide interaction with the DPC micelle (Connolly surface in gray) from Sybyl. The top view of the peptide on the surface of the micelle is depicted. The peptide is colored according to hydrophobic (orange) and hydrophilic (blue) residues, with the N-terminus to the right and C-terminus to the left (the same perspective as in panel A).

Figure 4

Plots of PBP 5 residue backbone N-H dynamic values. Error bars indicate standard deviations. (A) Plot of S² from the Modelfree calculation for each residue. The lower the S² value the greater amplitude of motion for the N–H vector. (B) Plot of effective correlation time (τ_e, ♦) and conformational exchange (R_ex, ■) from the Modelfree calculation for each residue that is described by the parameter. Data are only presented if the motional model for the residues is contained in these terms. The parameter τ_e describes fast motions on the ps time scale and R_ex describes conformational exchange on the ms time scale.

Figure 5

NOE interactions of PBP 5 anchor peptide protons to DPC micelle protons. The peptide atoms are colored according to the proton interactions with DPC. The atoms in black have no interaction with the micelle or the interactions overlap with the intra-peptide NOEs and can not be resolved. The peptide atoms where the carbon is one color and the hydrogen is another color indicates that those peptide protons are interacting with two colored regions of DPC and indicate with which regions the interactions occur. The structure of DPC is below the peptide sequence. The interacting regions are color coded according to resolved ¹H NMR signals that interact with the peptide.

Figure 6

Orientation of the anchor peptide in the membrane and its relationship to the surface domains of PBP 5. The arrow indicates the connection point between the anchor and the surface domains. The relative disposition of the surface and the membrane domains is likely, although it cannot be surmised from the work presented in this report.