NEDD8 Deamidation Inhibits Cullin RING Ligase Dynamics.

Cullin-RING ligases (CRLs) are a significant subset of Ubiquitin E3 ligases that regulate multiple cellular substrates involved in innate immunity, cytoskeleton modeling, and cell cycle. The glutamine deamidase Cycle inhibitory factor (Cif) from enteric bacteria inactivates CRLs to modulate these processes in the host cell. The covalent attachment of a Ubiquitin-like protein NEDD8 catalytically activates CRLs by driving conformational changes in the Cullin C-terminal domain (CTD). NEDDylation results in a shift from a compact to an open CTD conformation through non-covalent interactions between NEDD8 and the WHB subdomain of CTD, eliminating the latter's inhibitory interactions with the RING E3 ligase-Rbx1/2. It is unknown whether the non-covalent interactions are sufficient to stabilize Cullin CTD's catalytic conformation. We studied the dynamics of Cullin-CTD in the presence and absence of NEDD8 using atomistic molecular dynamics (MD) simulations. We uncovered that NEDD8 engages in non-covalent interactions with 4HB/αß subdomains in Cullin-CTD to promote open conformations. Cif deamidates glutamine 40 in NEDD8 to inhibit the conformational change in CRLs by an unknown mechanism. We investigated the effect of glutamine deamidation on NEDD8 and its interaction with the WHB subdomain post-NEDDylation using MD simulations and NMR spectroscopy. Our results suggest that deamidation creates a new intramolecular salt bridge in NEDD8 to destabilize the NEDD8/WHB complex and reduce CRL activity.

An "up" oriented methionine-aromatic structural motif in SUMO is critical for its stability and activity.

Protein structural bioinformatic analyses suggest preferential associations between methionine and aromatic amino acid residues in proteins. Ab initio energy calculations highlight a conformation-dependent stabilizing interaction between the interacting sulfur-aromatic molecular pair. However, the relevance of buried methionine-aromatic motifs to protein folding and function is relatively unexplored. The Small Ubiquitin-Like Modifier (SUMO) is a ß-grasp fold protein and a common posttranslational modifier that affects diverse cellular processes, including transcriptional regulation, chromatin remodeling, metabolic regulation, mitosis, and meiosis. SUMO is a member of the Ubiquitin-Like (UBL) protein family. Herein, we report that a highly conserved and buried methionine-phenylalanine motif is a unique signature of SUMO proteins but absent in other homologous UBL proteins. We also detect that a specific "up" conformation between the methionine-phenylalanine pair of interacting residues in SUMO is critical to its ß-grasp fold. The noncovalent interactions of SUMO with its ligands are dependent on the methionine-phenylalanine pair. MD simulations, NMR, and biophysical and biochemical studies suggest that perturbation of the methionine-aromatic motif disrupts native contacts, modulates noncovalent interactions, and attenuates SUMOylation activity. Our results highlight the importance of conserved orientations of Met-aromatic structural motifs inside a protein core for its structure and function.

Non-covalent Interaction With SUMO Enhances the Activity of Human Cytomegalovirus Protein IE1.

Viruses interact with the host cellular pathways to optimize cellular conditions for replication. The Human Cytomegalovirus (HCMV) Immediate-Early protein 1 (IE1) is the first viral protein to express during infection. It is a multifunctional and conditionally essential protein for HCMV infection. SUMO signaling regulates several cellular pathways that are also targets of IE1. Consequently, IE1 exploits SUMO signaling to regulate these pathways. The covalent interaction of IE1 and SUMO (IE1-SUMOylation) is well studied. However, the non-covalent interactions between SUMO and IE1 are unknown. We report two SUMO-Interacting Motifs (SIMs) in IE1, one at the end of the core domain and another in the C-terminal domain. NMR titrations showed that IE1-SIMs bind to SUMO1 but not SUMO2. Two critical functions of IE1 are inhibition of SUMOylation of Promyelocytic leukemia protein (PML) and transactivation of viral promoters. Although the non-covalent interaction of IE1 and SUMO is not involved in the inhibition of PML SUMOylation, it contributes to the transactivation activity. The transactivation activity of IE1 was previously correlated to its ability to inhibit PML SUMOylation. Our results suggest that transactivation and inhibition of PML SUMOylation are independent activities of IE1.

Stability of Begomoviral pathogenicity determinant ßC1 is modulated by mutually antagonistic SUMOylation and SIM interactions.

BACKGROUND: To successfully invade new hosts, plant viruses must break host resistance and be competent to move within and between plant cells. As a means, viral proteins known as pathogenicity determinants have evolved to coordinate a network of protein interactions. The ßC1 protein encoded by specific geminiviral satellites acts as a key pathogenicity determinant for this disease-causing family of plant viruses. Post-translational modifications (PTMs) such as ubiquitination and phosphorylation of the ßC1 protein have been shown to occur in diverse viruses. However, the relevance of these and other layers of PTMs in host-geminiviral interactions has not been fully understood. RESULTS: Here we identified the significance of a novel layer of PTMs in the ßC1 protein of Synedrella yellow vein clearing virus (SyYVCV), a newly identified member of the Begomovirus genus of Geminiviruses. This protein has conserved SUMOylation and SUMO-interacting motifs (SIMs), and we observed SUMOylation of SyYVCV ßC1 in host plants as a defensive strategy against ubiquitin-mediated degradation. Counteracting this, SIMs encoded in ßC1 mediate the degradation of ßC1; however, both these PTMs are essential for the function of ßC1 protein since SIM and SUMOylation motif mutants failed to promote pathogenicity and viral replication in vivo. SUMOylation in different motifs of ßC1 led to functionally distinct outcomes, regulating the stability and function of the ßC1 protein, as well as increased global SUMOylation of host proteins. CONCLUSION: Our results indicate the presence of a novel mechanism mediating a fine balance between defence and counter-defence in which a SIM site is competitively sought for degradation and, as a counter-defence, ßC1 undergoes SUMOylation to escape from its degradation.

Amide temperature coefficients in characterizing the allosteric effects of ligand binding on local stability in proteins.

Proteins can stabilize upon binding a ligand. Due to allosteric effects, the changes in stability can occur at regions far from the protein:ligand interface. Efficient methods to measure the changes in local stability upon ligand binding will be useful to understand allostery and may be helpful in protein engineering. In this work, we suggest the measurement of backbone amide temperature coefficients to probe the effect of ligand binding on the local stability of ß-sheet rich proteins. The method was applied for two protein:ligand complexes with different binding affinities. The protein includes a beta-sheet network connected by hydrogen bonds. The measured temperature coefficient data captured the stabilizing effect of ligand binding, which propagated across the beta-sheet network of the protein. Intriguingly, the impact on the local and global stability of the protein was proportional to the strength of protein:ligand interaction.

Casein kinase-2-mediated phosphorylation increases the SUMO-dependent activity of the cytomegalovirus transactivator IE2.

Many viral factors manipulate the host post-translational modification (PTM) machinery for efficient viral replication. In particular, phosphorylation and SUMOylation can distinctly regulate the activity of the human cytomegalovirus (HCMV) transactivator immediate early 2 (IE2). However, the molecular mechanism of this process is unknown. Using various structural, biochemical, and cell-based approaches, here we uncovered that IE2 exploits a cross-talk between phosphorylation and SUMOylation. A scan for small ubiquitin-like modifier (SUMO)-interacting motifs (SIMs) revealed two SIMs in IE2, and a real-time SUMOylation assay indicated that the N-terminal SIM (IE2-SIM1) enhances IE2 SUMOylation up to 4-fold. Kinetic analysis and structural studies disclosed that IE2 is a SUMO cis-E3 ligase. We also found that two putative casein kinase 2 (CK2) sites adjacent to IE2-SIM1 are phosphorylated in vitro and in cells. The phosphorylation drastically increased IE2-SUMO affinity, IE2 SUMOylation, and cis-E3 activity of IE2. Additional salt bridges between the phosphoserines and SUMO accounted for the increased IE2-SUMO affinity. Phosphorylation also enhanced the SUMO-dependent transactivation activity and auto-repression activity of IE2. Together, our findings highlight a novel mechanism whereby SUMOylation and phosphorylation of the viral cis-E3 ligase and transactivator protein IE2 work in tandem to enable transcriptional regulation of viral gene.

A conserved and buried edge-to-face aromatic interaction in small ubiquitin-like modifier (SUMO) has a role in SUMO stability and function.

Aromatic amino acids buried at a protein's core are often involved in mutual paired interactions. Ab initio energy calculations have highlighted that the conformational orientations and the effects of substitutions are important for stable aromatic interactions among aromatic rings, but studies in the context of a protein's fold and function are elusive. Small ubiquitin-like modifier (SUMO) is a common post-translational modifier that affects diverse cellular processes. Here, we report that a highly conserved aromatic triad of three amino acids, Phe36-Tyr51-Phe64, is a unique SUMO signature that is absent in other ubiquitin-like homologous folds. We found that a specific edge-to-face conformation between the Tyr51-Phe64 pair of interacting aromatics is vital to the fold and stability of SUMO. Moreover, the noncovalent binding of SUMO-interacting motif (SIM) at the SUMO surface was critically dependent on the paired aromatic interactions buried at the core. NMR structural studies revealed that perturbation of the Tyr51-Phe64 conformation disrupts several long-range tertiary contacts in SUMO, leading to a heterogeneous and dynamic protein with attenuated SUMOylation both in vitro and in cells. A subtle perturbation of the edge-to-face conformation by a Tyr to Phe substitution significantly decreased stability, SUMO/SIM affinity, and the rate of SUMOylation. Our results highlight that absolute co-conservation of specific aromatic pairs inside the SUMO protein core has a role in its stability and function.

Meta effect of absorption energy in donor-acceptor substituted benzenoids: a computational study of its dependence on acceptor strength, solvent polarity, and conjugation length.

The present work focuses on theoretical understanding of electronic absorption energies of N,N-dimethylaniline with different ortho-, meta-, and para-substituted acceptor groups. The meta isomers exhibit the lowest absorption energy compared to the ortho and para derivatives. This unusual behavior of absorption energies of the meta isomers is related to the "meta effect" well-known in organic photochemical reactions. The meta effect of absorption energy of the derivatives is found to depend on the strength of acceptors, solvent polarity, and conjugation length. The meta derivatives with strong acceptor groups generally exhibit the lowest absorption energy over the other isomers irrespective of solvent polarity. However, the meta isomers with weak acceptor groups exhibit the meta effect only in highly polar solvents. The trend of the lowest absorption energies of the meta isomers is observed to change if the acceptor group is bridged through π conjugation unit (n) with the core moiety. The normal pattern of absorption energy that is the para isomer is of lowest energy is observed to occur for the derivatives where the repeated conjugation units (n) are between 2 and 4. The normal pattern of absorption energy is continued to observe from n > 4 for all the derivatives.