1. Volume 24, Issue 12, Pages 2127-2137 (December 2016)
Structure of the NPr:EINNtr Complex: Mechanism for Specificity in Paralogous Phosphotransferase Systems 
Madeleine Strickland, Ann Marie Stanley, Guangshun Wang, Istvan Botos, Charles D. Schwieters, Susan K. Buchanan, Alan Peterkofsky, Nico Tjandra 
Structure 
Volume 24, Issue 12, Pages (December 2016)
DOI: /j.str
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2. Structure 2016 24, 2127-2137DOI: (10.1016/j.str.2016.10.007)
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3. Figure 1
Retention of Specificity of EINtr and EIsugar by N-Terminal Domains
(A) The specificity of the interactions was demonstrated in a Biacore binding experiment. Surface plasmon resonance response for His6-EINNtr-WT and His6-EINsugar that were immobilized on sensor chips are shown in black and red lines, respectively. Vertical arrows indicate the beginning of the time interval for flow of the indicated ligand over the chip.
(B) The specificity was demonstrated by chromatography on a Superose 12 gel filtration column, followed by SDS-PAGE. Upper panel: mixing of EINNtr−H356Q with HPr results in no indication of binding. Lower panel: mixing of EINNtr−H356Q with NPr instead gives a clear coelution at a higher molecular weight, indicating binding.
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4. Figure 2 Refinement of the NPr Structure in the Free Form
(A) Residue-specific backbone amide residual dipolar couplings (RDCs) obtained using Pf1 filamentous phage as an alignment medium (circles) and calculated RDCs based on the refined structure (black line).
(B) Observed RDCs versus calculated RDCs.
(C) Residue-specific amide backbone proton pseudocontact shifts (PCS) obtained by calculating the difference between Lu-M8-NPrE45C (diamagnetic) and Yb-M8-NPrE45C (paramagnetic) chemical shifts (circles) and back-calculated PCS based on the refined structure (black line).
(D) Observed versus calculated proton PCS.
(E) Bundle of the 20 lowest energy free NPr structures from the Xplor-NIH refinement, selected from a calculation of 200 structures. The N and C termini, the α helices (red), and β sheets (yellow) are highlighted, with loops in green.
(F) Comparison of the lowest energy free NPr (blue) and free HPr (red) structures (HPr PDB: 1POH; Jia et al., 1993). Active-site histidines H15 (HPr) and H16 (NPr) are shown using sticks.
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5. Figure 3
Chemical Shift Perturbations of EINNtr and NPr upon Formation of the NPr:EINNtr Complex
(A and B) NMR chemical shift differences (Δδ, ppm) between the free and complexed forms of EINNtr (A) and NPr (B). Binding-site residues are indicated by large Δδ values, or missing Δδ values in residues that are dynamic. Regions used as ambiguous distance restraints in the docking of the NPr:EINNtr complex are highlighted in blue, with the active-site residues H356Q of EINNtr and H16 of NPr indicated with red arrows.
(C) The 1H/15N-HSQC NMR spectrum of 2H/13C/15N-EINNtr in the presence (red) and absence (black) of 2H-NPr. Residues that undergo a large change upon complexation are highlighted with blue lines.
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6. Figure 4
RDC Data for the NPr:EINNtr Complex Fit against the EINNtr Crystal Structure
(A and B) Observed backbone amide 1H/15N-RDCs collected for 2H/13C/15N-EINNtr in the presence of 2H-NPr vs (A) calculated RDCs for the EINNtr crystal structure and (B) HPr:EINsugar structure (PDB: 3EZA; Garrett et al., 1999).
(C) X-ray crystal structure of EINNtr, with residues used for RDC fitting of individual domains shown in different colors (C-terminal helix in green, α/β domain in red, the two halves of the α domain in yellow and purple, loops in gray, and regions without electron density shown with dotted lines).
(D–G) Observed RDCs as in (A) versus calculated RDCs for each individual domain of the EINNtr crystal structure (colors as in C).
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7. Figure 5
Docking and Refinement of the NPr:EINNtr NMR Complex Structure
(A and B) Residue-specific backbone amide residual dipolar couplings (RDCs) obtained using Pf1 filamentous phage as an alignment medium (filled circles) and calculated RDCs based on the refined structure (black line) for EINNtr (A) and NPr (B) measured for the NPr:EINNtr complex.
(C) Observed versus calculated RDCs for EINNtr (turquoise) and NPr (red).
(D and E) Residue-specific amide backbone proton pseudocontact shifts (PCS) obtained by calculating the difference between Lu-M8-NPrE45C:EINNtr (diamagnetic) and Yb-M8-NPrE45C:EINNtr (paramagnetic) chemical shifts (filled circles) and calculated PCS based on the refined structure (black line) for EINNtr (D) and NPr (E).
(F) Observed versus calculated proton PCS for EINNtr (turquoise) and NPr (red).
(G) EINNtr X-ray crystal structure with α, α/β, and C-terminal domains highlighted.
(H) Lowest energy structure of the NPr:EINNtr complex (NPr not shown).
(I) HPr:EINsugar complex (HPr not shown) (PDB: 3EZA; Garrett et al., 1999).
(J) Observed SAXS data, ln(I), for the NPr:EINNtr complex (yellow circles) shown with the curve back-calculated from the lowest energy NPr:EINNtr complex structure in black.
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8. Figure 6
Comparison of the NPr:EINNtr and HPr:EINsugar Complex Structures and Active Sites
(A) Lowest energy structure of the NPr:EINNtr complex calculated using Xplor-NIH, with EINNtr and NPr in yellow and red, respectively, both in cartoon and surface representation.
(B) Lowest energy structure of the HPr:EINsugar complex (PDB: 3EZA; Garrett et al., 1999), with EINsugar and HPr in green and blue, respectively, both in cartoon and surface representation.
(C and D) Active sites of NPr:EINNtr (C) and HPr:EINsugar (D) with residues conserved between the two complexes shown in sticks. NPr and HPr residues are represented in red and blue sticks, respectively, while histidines that participate in phosphoryl transfer are highlighted with stars. For the NPr:EINNtr structure, the mutated H356Q glutamine residue is shown.
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9. Figure 7
Similarities between Binding-Site Residues for HPr:EINsugar and NPr:EINNtr
(A) Alignment of NPr with HPr using EMBOSS Needle, with the active-site residue highlighted with a yellow background, and binding residues highlighted with stars. NPr:EINNtr and HPr:EINsugar binding regions are indicated using green and cyan lines, respectively. The degree of similarity between the sequences are indicated with vertical black lines (identical), one (low level of similarity) or two (high similarity) dots.
(B) Using the adaptive Poisson-Boltzmann solver (APBS), the electrostatics of the four proteins were calculated and are displayed on a surface representation of the structures (HPr and EINsugar are from PDB: 3EZA (Garrett et al., 1999). NPr and EINsugar are both negatively charged, while EINNtr and HPr are positively charged in the binding site regions, explaining the lack of cross-reactivity. Binding site residues are highlighted.
(C) Coloring as in (A) for EINNtr and EINsugar binding sites.
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10. Figure 8 Effectors of Paralogous Phosphotransferase Systems
The upper scheme depicts the PTS sugar pathway, which is responsible for sugar phosphorylation and import, while the lower scheme shows the PTSNtr. ∼P denotes the high-energy phosphoryl moiety (originating from phosphoenolpyruvate) that can be reversibly transferred between the various proteins (double-headed arrows). While phosphotransfer from EIs to HPr or NPr shows pathway specificity, those between HPr or NPr to EIIA proteins exhibit crosstalk. The specific regulatory actions associated with indicated proteins are illustrated. Arrows indicate positive effects, while blunt-end lines indicate inhibitory effects.
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