Fig 1.
Constructs for analyzing the AIMP3-LmnA interaction.
A. Primary structure difference between mature LmnA and LmnC. B. Recombinant protein constructs for interaction analysis.
Fig 2.
Sequence coverage for proteolytic peptides (5–30 aa in length) common to free His-Tev-AIMP3 and His-Tev-AIMP3 in complex (Top); and free His-Strep-TrxA-LmnA and His-Strep-TrxA-LmnA in complex (bottom).
Peptides containing less than 5 or more than 30 amino acids are not considered, due to increased ambiguity and poor sequence localization. The displayed segments cover 100% of the sequences based on the common segments.
Fig 3.
HDX heat map for deuterium uptake by free His-Tev-AIMP3.
The His-Tev tag sequence is in grey. The deuteration level percentage is calculated by dividing the observed deuterium uptake by the total number of amide hydrogens (not counting proline) in that segment. For each peptide, the calculated deuteration level for each HDX incubation period (top left, proceeding from top to bottom: incubation periods of 0.5, 1, 2, 4, 8, 15, 30, 60, 120, and 240 min) is mapped onto the sequence. Secondary structure is noted on top of the sequence (PDB 2UZ8) [53]. Alpha helices and beta strands are numbered in order from N to C terminus.
Fig 4.
HDX heat map for deuterium uptake by free His-Strep-TrxA-LmnA.
The His-Strep-TrxA tag sequence is in gray. The deuteration level percentage is calculated as for AIMP3.
Fig 5.
H/D exchange results for free and complexed AIMP3 with LmnA.
For each of the proteolytic peptides common to free and bound AIMP3, the relative D-uptake change for AIMP3 on binding to LmnA (ARDD) is calculated as described by Eq 1. Peptide regions with significant deuterium uptake differences are mapped onto the crystal structure. Top: AIMP3 shows decreases in D-uptake for segments 91–96 and 134–152 upon binding to LmnA, consistent with the binding interface between AIMP3 and LmnA. Bottom: ARDD mapped onto the crystal structure (PDB 2UZ8). Note that segments 91–96 and 134–139 (red circle) are spatially close to each other.
Fig 6.
Deuterium uptake profiles (data points) and maximum-entropy fits (smooth curves [54]) vs. H/D exchange period (log10 scale) for selected segments of free and complexed AIMP3.
Segment 91–96 of putative binding Interface I exhibits a significant decrease in D-uptake upon forming the complex. Segments 127–133 and 158–171 constitute putative Interface II, but show no change in D-uptake. Significant decreases are also observed for segments 134–139 and 143–152, thereby defining the AIMP3 binding surface to LmnA.
Fig 7.
H/D exchange results for free and complexed LmnA with AIMP3.
ARDD for LmnA is calculated as for AIMP3. Top: LmnA shows decreased D-uptake for segment 203–209 (641–647) upon binding AIMP3. Bottom: Deuterium uptake profiles (data points) and maximum-entropy fits (smooth curves [54]) vs. H/D exchange period (log10 scale) for selected segments of free and complexed LmnA. Left: Selected peptide 64–73, representing peptides in the His-Strep-TrxA tag, shows unaltered deuteration level. Right: Peptide 203–209 (641–647) shows significantly decreased D-uptake upon binding AIMP3.
Fig 8.
Potential binding regions mapped onto the crystal structure of AIMP3.
Upper left: Interface I in yellow, including residues Arg50, Thr68, Lys75, Ala91, Gln94, Gln95, Glu98, and Asp119. Upper right: Segment 91–96 with significant decrease in D-uptake, overlapping with putative binding interface I. Lower left: Putative binding Interface II in green, including residues Glu125, Val128, Tyr129, Tyr133, Leu162, Arg166, and Phe186, does not exhibit any D-uptake change. Lower right: Novel binding site including segments N134FTLAD139, and L143YYGLHRFIV152. Color codes for the residues represent Average Relative D-uptake difference (see Eq 1).