NMR characterization of conformational fluctuations and noncovalent interactions of SUMO protein from Drosophila melanogaster (dSmt3).

Structural heterogeneity in the native-state ensemble of dSmt3, the only small ubiquitin-like modifier (SUMO) in Drosophila melanogaster, was investigated and compared with its human homologue SUMO1. Temperature dependence of amide proton's chemical shift was studied to identify amino acids possessing alternative structural conformations in the native state. Effect of small concentration of denaturant (1M urea) on this population was also monitored to assess the ruggedness of near-native energy landscape. Owing to presence of many such amino acids, especially in the β2 -loop-α region, the native state of dSmt3 seems more flexible in comparison to SUMO1. Information about backbone dynamics in ns-ps timescale was quantified from the measurement of 15 N-relaxation experiments. Furthermore, the noncovalent interaction of dSmt3 and SUMO1 with Daxx12 (Daxx729 DPEEIIVLSDSD740 ), a [V/I]-X-[V/I]-[V/I]-based SUMO interaction motif, was characterized using Bio-layer Interferometery and NMR spectroscopy. Daxx12 fits itself in the groove formed by β2 -loop-α structural region in both dSmt3 and SUMO1, but the binding is stronger with the former. Flexibility of β2 -loop-α region in dSmt3 is suspected to assist its interaction with Daxx12. Our results highlight the role of native-state flexibility in assisting noncovalent interactions of SUMO proteins especially in organisms where a single SUMO isoform has to tackle multiple substrates single handedly.