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

Abstract

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 15N‐relaxation experiments. Furthermore, the noncovalent interaction of dSmt3 and SUMO1 with Daxx12 (Daxx729DPEEIIVLSDSD740), 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.

Covalent versus Noncovalent Binding of Ruthenium η6‐p‐Cymene Complexes to Zinc‐Finger Protein NCp7

“…Thec oncentration-dependent shift of NMR signals indicates af ast exchange of NCp7 upon Ru-4 binding, which is consistent with noncovalent binding of Ru-4 to the protein. [16] In addition, the graduals hift of the N e Hs ignal of the tryptophan residue (10.3/128.5 ppm in Figure S4A) suggests that the aromatic indole ring participates in the noncovalent interaction with Ru-4.T he overall signal shifts of NCp7a re basically small,i ndicating only minimal structural alteration of NCp7 upon binding Ru-4,w hichi si nl ine with the result of CD measurements. Different from Ru-4,t he reactiono fRu-5 led to as low disappearance of some signals in at ime-dependent manner ( Figure 4B and S4B).…”

“…The binding of Ru‐4 caused a shift of the signals, and peaks moved to a different extent upon Ru‐4 binding (Figure A and S4A). The concentration‐dependent shift of NMR signals indicates a fast exchange of NCp7 upon Ru‐4 binding, which is consistent with noncovalent binding of Ru‐4 to the protein . In addition, the gradual shift of the N ϵ H signal of the tryptophan residue (10.3/128.5 ppm in Figure S4A) suggests that the aromatic indole ring participates in the noncovalent interaction with Ru‐4 .…”

Conformational Dynamics and metal-ion interactions in human small Ubiquitin-like modifier (SUMO2)

“…Looking the native structure of SUMO2 for explaining its more stability over SUMO1 which is more aggregation prone in presence or absence of metal ions, SUMO2 shares a close similarity to dSmt3 (SUMO from Drosophilla melanogaster[41]) as both proteins were found to contain conformationally flexible amino acids at equivalent locations in the loop-α 1 region ( Fig. 5d ).…”

“…The Daxx12-H (DPEEIIVLSDSD) was chemically synthesized in a solid phase peptide synthesizer (PS3; Protein Technologies, Medford, MA) as described previously [28]. the locking and chemical shift referencing.…”

Characterizing the Conformational Dynamics of Human SUMO2: Insights into its Interaction with Metal Ions and SIMs

“…Covalent tagging of proteins by SUMO proteins, also known as SUMOylation, is a crucial post-translational modification. [1][2] It is mechanistically similar to ubiquitination and involves the covalent isopeptide bond linkage to the Lysine residue in the ψ-K-x-D/E motif, present in the target molecules. [3] SUMOylation involves covalent or non-covalent interactions yield distinct consequences.…”