Structure-Based Insights into the Dynamics and Function of Two-Domain SlpA from Escherichia coli

Abstract

SlpA (SlyD-like protein A) comprises two domains, a FK506 binding domain (FKBP fold) of moderate prolyl cis/trans-isomerase activity and an inserted in flap (IF) domain that hosts its chaperone activity. Here we present the nuclear magnetic resonance (NMR) solution structure of apo Escherichia coli SlpA determined by NMR that mirrors the structural properties seen for various SlyD homologues. Crucial structural differences in side-chain orientation arise for F37, which points directly into the hydrophobic core of the active site. It forms a prominent aromatic stacking with F15, one of the key residues for PPIase activity, thus giving a possible explanation for the inherently low PPIase activity of SlpA. The IF domain reveals the highest stability within the FKBP-IF protein family, most likely arising from an aromatic cluster formed by four phenylalanine residues. Both the thermodynamic stability and the PPIase and chaperone activity let us speculate that SlpA is a backup system for homologous bacterial systems under unfavorable conditions.

Multicopy Suppressor Analysis of Strains Lacking Cytoplasmic Peptidyl-Prolyl cis/trans Isomerases Identifies Three New PPIase Activities in Escherichia coli That Includes the DksA Transcription Factor

“…Nevertheless, the relative PPIase activity of DksA and FkpB are comparable ( Figure 6 A). The catalytic efficiency of DksA turns out to be k cat / K M 0.7 × 10 3 M −1 s −1 as compared to 1.25 × 10 3 M −1 s −1 for FkpB, which is comparable to previously reported activity of FkpB [ 21 ]. The relatively weaker PPIase activity of these multicopy suppressing factors can explain why DksA, MetL and Cmk have not been previously identified as PPI enzymes.…”

“…Furthermore, this PPIase activity of DksA, Cmk and MetL could be inhibited by FK506 establishing them as bona fide PPIs. It should be noted that, although the PPIase activity of DksA is 1000-fold less than classical PPIs such as FklB [ k cat / K M 0.7 x 10 3 M −1 s −1 for DksA vs. k cat / K M 10 6 M −1 s −1 for FklB ( Figure 6 A)], it is still comparable to the PPIase activity of FkpB—a well characterized PPI with the low PPI activity with several substrates [ 1 , 21 ]. However, more experiments will be required to identify the binding affinity of these proteins with FK506, the contact regions where FK506 can bind and exact residues involved in catalysis.…”

Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers

“…4A,B). Both parameters refer to motions of the 1 H- 15 N bond vector on the picosecond to nanosecond time scale providing general information about backbone flexibility 57 . Generally, hetNOE values are high and RMSF values are low consistently over the sequence for both the Lys11-, and the Lys27-linkage type underlying the stability of the β-grasp Ub fold (Fig.…”

Bacterial Chaperone Domain Insertions Convert Human FKBP12 into an Excellent Protein-Folding Catalyst—A Structural and Functional Analysis

“…The proteins SlpA and FKBP18 naturally contain IF domains. The excision of the IF domain decreases the midpoint of the GdmCl-induced unfolding transition from 1.7 M to 1.4 M GdmCl (Table 1) [25], which corresponds to a destabilization by 5.8 kJ mol −1 . In the case of FKBP18, the excision of the IF domain increases the midpoint of the GdmCl-induced unfolding transition from 3.6 M to 3.8 M GdmCl, but the cooperativity is decreased from 10.0 to 7.4 kJ mol −1 M −1 , corresponding to a decrease in stability by 7.8 kJ mol −1 , in line with experiments of SlyD from T. thermophilus [17].…”

“…In the designed chimeric FKBP12 variants, two different bacterial chaperone domains were inserted into the flap loop near the catalytic site. These domains were the chaperone domains of the E. coli protein SlpA [15,24,25] and of FKBP18 from Thermococcus sp. (Figure 1A).…”