Fig 1.
Figure based on PDB entry 2KFW, which is a solution NMR structure of full-length SlyD from E. coli. Linker region in grey. Residues mutated in this study are indicated.
Table 1.
Primers used in this study1.
Fig 2.
Peptide screen analyses demonstrate selective binding of SlyD to peptides with a positive net charge.
A) Screen covering all E. coli Tat signal peptides and HiPIP, as indicated. See paragraph “peptide spot arrays” in the Methods section for technical details, and supplementary S2 Table for a list of the sequences. Left: Original autoradiogram showing the signals of 35S-labeled SlyD. Right: Schematic diagram, showing the peptides with positive net charge in indicated colors. B) Analysis of SlyD-binding to indicated amino acids in a glycine environment. C) Analysis of SlyD-binding to twin-arginines in an indicated uncharged environment. Left: Original data, Right: Quantification of the signals, normalized to the signal obtained with glycine, with bars in the following color code: brown, aromatic; yellow, aliphatic; green polar; blue, glycine (used for normalization).
Fig 3.
SlyD binds wild type HiPIP precursor (RR-HiPIP) with higher affinity than the HiPIP precursor with an RR>KK or RR>QQ exchange.
ITC analysis of SlyD-binding to RR-HiPIP, KK-HiPIP, and QQ-HiPIP. KD values are indicated. See Methods for details. The signal peptides of the analyzed proteins are shown at the top of the figure, with the two potential SlyD-binding regions as identified in the peptide scan (Fig 2) and the twin-arginines underlined, and residues colored according to their properties (blue: positive; red: negative; ocher: hydrophobic; black: all other).
Fig 4.
Mutation of charged residues have less effect on substrate affinity than mutations of the active site phenylalanines.
ITC analyses of mutated SlyD variants A) WebLogo [44] analysis of an alignment of SlyD sequences from a wide range or organisms (see S1 Fig and S1 Table). The alignment was done using Clustal-Omega [43]. B) SDS PAGE analysis of purified SlyD variants (Coomassie stain); C) ITC titration of indicated SlyD variants. See S3 Table for complete data set.
Fig 5.
Two of the SlyD variants with exchanged negative charges are significantly affected in HiPIP signal peptide-binding.
Plots of KD, ΔH, and ΔS for the SlyD variants with indicated exchanged negatively charged residues. The data from the wild type and the Y68S variant were included as negative controls. See text for details.
Fig 6.
A highly negative IF domain surface is conserved among SlyD orthologs in archaea and bacteria.
A) pI values of SlyD IF domains from species of indicated phyla or classes, in comparison to the overall pI of SlyD and the average pI of the whole proteome of the respective organism (see Methods for details). B) Electrostatic surface charge characteristics of SlyD IF domains from T. thermophilus and E. coli, as examples of typical acidic SlyD IF domains, and from B. bacteriovorus, the only to us known example of a “switched” alkaline SlyD IF domain. Only the IF domains are shown for clarity reasons. See text for details.
Fig 7.
Model for the binding of helical signal peptides by SlyD.
A) The SlyD-bound protein E of phage ΦX174 (yellow) aligns its α-helix in a way that uses the hydrophobic pocket and adjacent negatively charged surface for the interaction (PDB 8G02). Positively charged residues of protein E are shown and colored in blue. The electrostatic potential is indicated in the left image (red: negative, blue: positive), whereas the right image facilitates the recognition of the secondary and tertiary structures that are involved. The IF and FKBP domains are labeled and their relative orientations are different from the structure shown in Fig 1, which is due to the flexible linker region that connects both domains. Note that the bound region of protein E resembles the twin-arginine motif region of HiPIP that is bound by SlyD. B) Comparison of SlyD IF domain structures as found when no substrate is bound (closed; PDB 2K8I) or when the helical substrate protein E is bound (open; substrate hidden; PDB 8G02). A conformational change of two beta-strands that opens access to the hydrophobic pocket from a vertically aligned α-helix is induced by binding of protein E to SlyD. The positions of the hydrophobic pocket residues F84 and F96 are shown, as well as the position of the D101 residue that is at the tip beta sheet that opens upon substrate interaction. Note that the orientation of F96 changes from the closed to the open state. C) Schematic binding modes for helical peptides to the IF domain. An initial electrostatic interaction induces a conformational change that permits binding of the hydrophobic residues by SlyD.