1. X-Ray Crystallography and Electron Microscopy of Cross- and Multi-Module Nonribosomal Peptide Synthetase Proteins Reveal a Flexible Architecture 
Michael J. Tarry, Asfarul S. Haque, Khanh Huy Bui, T. Martin Schmeing 
Structure 
Volume 25, Issue 5, Pages e4 (May 2017)
DOI: /j.str
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2. Structure 2017 25, 783-793.e4DOI: (10.1016/j.str.2017.03.014)
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3. Figure 1 Bacillibactin Synthesis
(A) DhbE and DhbB are stand-alone A and aryl carrier protein domains responsible for the activation and transfer of 2,3-dihydroxybenzoate (DHB). DhbF adds glycine and threonine to the growing peptide chain. An MLP found in the NRPS cluster may be required for activity of one or both A domains. The Te domain is responsible for cyclization of three DHB-Gly-Thr peptides and release of bacillibactin.
(B) Structure of bacillibactin.
The constructs used for X-ray crystallography (black dashed outline) and electron microscopy (EM) (gray dashed outlines) are indicated.
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4. Figure 2
Adenylation Activity of DhbFA1-PCP1-C2 Requires the MLP and Can Be Inhibited by an AVS
(A) The adenylation activity of the A1 domain of DhbFA1-PCP1-C2 (blue), DhbFA1-PCP1-C2-MLP (red), and DhbFA1-PCP1-C2-MLP inhibited with Gly-AVS. Samples were pre-incubated with either 1.25-fold (green, trial 1) or 6.25-fold (purple, trial 2) molar excess of Gly-AVS and assessed using the pyrophosphate production assay. Note that the inhibition is caused by AVS covalently bound to the enzyme, not excess free AVS, so if full covalent modification is achieved by both procedures, inhibition should be identical, as observed. Error bars signify SDs from duplicate experiments.
(B) Deconvoluted mass spectra of DhbFA1-PCP1-C2-MLP (upper panel) and DhbFA1-PCP1-C2-MLP after incubation with a 1.25-fold molar excess of Gly-AVS (lower panel) shows essentially full conversion to the dead-end Gly-AVS-modified DhbFA1-PCP1-C2-MLP. The expected molecular masses of DhbFA1-PCP1-C2 and Gly-AVS-modified DhbFA1-PCP1-C2 are shown with the schematics. No protein with the mass of DhbFA1-PCP1-C2 lacking a phosphopantetheine (121,204.3 Da, arrow) was detected in the sample, indicating that “apo” DhbFA1-PCP1-C2-MLP was not the source of residual adenylation activity.
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5. Figure 3 Structure of DhbFA1-PCP1-C2-MLP
(A) Cartoon representation of the overall DhbFA1-PCP1-C2-MLP structure. Missing linker regions between domains in the structure are indicated by dashed gray lines. The Gly-AVS-bound PPE arm is shown in stick form with the S999 attachment residue indicated.
(B) The Gly-AVS covalently attached to the PPE arm, bound at the A-domain active site with Fo-Fc density map shown in green, contoured at 3σ, carved at 8 Å.
(C) Detailed view of the interdomain and intermodular interactions. Intermodular contacts are mediated exclusively by the PCP domain.
(D) MLP binding the Acore.
See also Figures S1, S2, and S7.
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6. Figure 4
MLP Induces Conformational Changes in DhbFA1 and is Required for Substrate Binding
(A) Size-exclusion chromatography of purified DhbFA1 (blue), DhbFA1-MLP (purple), DhbFA1core (red), and DhbFA1core-MLP (black). Elution positions of molecular weight standards (kDa) are shown above the trace. Inset is a native PAGE gel of purified DhbFA1, DhbFA1-MLP, DhbFA1core, and DhbFA1core-MLP, with molecular weight standards (kDa). Elution volumes and apparent molecular weights are reported in Table S1.
(B) ITC data for MLP binding to DhbFA1. The upper panel shows the raw heat of injection. The number of binding sites and KD were determined by fitting the integrated isotherm (lower panel) to a single-site binding model.
(C–E) ITC data for binding of glycine (C), ATP (D), and Gly-AVS (E) to DhbFA1-MLP (black) or DhbFA1 (red). The KD for binding to DhbFA1-MLP was determined using a single-site binding model with the number of binding sites fixed at 1. See also Figure S3.
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7. Figure 5 Two-Dimensional Class Averages of Dimodular DhbF
Selected Two-Dimensional Class Averages of DhbFΔTe-MLP are shown.
See also Figures S4–S6.
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8. Figure 6
Refined Three-Dimensional Envelopes Fit with Models of Dimodular DhbF
See also Figures S4–S6.
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