Ranabir Das

Allosteric Activation of E2-RING Finger-Mediated Ubiquitylation by a Structurally Defined Specific E2-Binding Region of gp78

The activity of RING finger ubiquitin ligases (E3) is dependent on their ability to facilitate transfer of ubiquitin from ubiquitin-conjugating enzymes (E2) to substrates. The G2BR domain within the E3 gp78 binds selectively and with high affinity to the E2 Ube2g2. Through structural and functional analyses, we determine that this occurs on a region of Ube2g2 distinct from binding sites for ubiquitin-activating enzyme (E1) and RING fingers. Binding to the G2BR results in conformational changes in Ube2g2 that affect ubiquitin loading. The Ube2g2:G2BR interaction also causes an approximately 50-fold increase in affinity between the E2 and RING finger. This results in markedly increased ubiquitylation by Ube2g2 and the gp78 RING finger. The significance of this G2BR effect is underscored by enhanced ubiquitylation observed when Ube2g2 is paired with other RING finger E3s. These findings uncover a mechanism whereby allosteric effects on an E2 enhance E2-RING finger interactions and, consequently, ubiquitylation.

Allosteric regulation of E2:E3 interactions promote a processive ubiquitination machine

RING finger proteins constitute the large majority of ubiquitin ligases (E3s) and function by interacting with ubiquitin‐conjugating enzymes (E2s) charged with ubiquitin. How low‐affinity RING–E2 interactions result in highly processive substrate ubiquitination is largely unknown. The RING E3, gp78, represents an excellent model to study this process. gp78 includes a high‐affinity secondary binding region for its cognate E2, Ube2g2, the G2BR. The G2BR allosterically enhances RING:Ube2g2 binding and ubiquitination. Structural analysis of the RING:Ube2g2:G2BR complex reveals that a G2BR‐induced conformational effect at the RING:Ube2g2 interface is necessary for enhanced binding of RING to Ube2g2 or Ube2g2 conjugated to Ub. This conformational effect and a key ternary interaction with conjugated ubiquitin are required for ubiquitin transfer. Moreover, RING:Ube2g2 binding induces a second allosteric effect, disrupting Ube2g2:G2BR contacts, decreasing affinity and facilitating E2 exchange. Thus, gp78 is a ubiquitination machine where multiple E2‐binding sites coordinately facilitate processive ubiquitination.

Functional evaluation of BRCA2 variants mapping to the PALB2 binding and C-terminal DNA binding domains using a mouse ES cell–based assay

Single-nucleotide substitutions and small in-frame insertions or deletions identified in human breast cancer susceptibility genes BRCA1 and BRCA2 are frequently classified as variants of unknown clinical significance (VUS) due to the availability of very limited information about their functional consequences. Such variants can most reliably be classified as pathogenic or non-pathogenic based on the data of their co-segregation with breast cancer in affected families and/or their co-occurrence with a pathogenic mutation. Biological assays that examine the effect of variants on protein function can provide important information that can be used in conjunction with available familial data to determine the pathogenicity of VUS. In this report, we have used a previously described mouse embryonic stem (mES) cell-based functional assay to characterize eight BRCA2 VUS that affect highly conserved amino acid residues and map to the N-terminal PALB2-binding or the C-terminal DNA-binding domains. For several of these variants, very limited co-segregation information is available, making it difficult to determine their pathogenicity. Based on their ability to rescue the lethality of Brca2-deficient mES cells and their effect on sensitivity to DNA-damaging agents, homologous recombination and genomic integrity, we have classified these variants as pathogenic or non-pathogenic. In addition, we have used homology-based modeling as a predictive tool to assess the effect of some of these variants on the structural integrity of the C-terminal DNA-binding domain and also generated a knock-in mouse model to analyze the physiological significance of a residue reported to be essential for the interaction of BRCA2 with meiosis-specific recombinase, DMC1.

A comprehensive functional characterization of BRCA2 variants associated with Fanconi anemia using mouse ES cell–based assay

Biallelic mutations in the human breast cancer susceptibility gene, BRCA2, are associated with Fanconi anemia, implying that some persons who inherit 2 deleterious variants of BRCA2 are able to survive even though it is well established that BRCA2 is indispensable for viability in mice. One such variant, IVS7 + 2T > G, results in premature protein truncation because of skipping of exon 7. Surprisingly, the persons who are either IVS7 + 2T > G homozygous or compound heterozygous are born alive but die of malignancy associated with Fanconi anemia. Using a mouse embryonic stem cell-based functional assay, we found that the IVS7 + 2T > G allele produces an alternatively spliced transcript lacking exons 4-7, encoding an in-frame BRCA2 protein with an internal deletion of 105 amino acids (BRCA2(Δ105)). We demonstrate that BRCA2(Δ105) is proficient in homologous recombination-mediated DNA repair as measured by different functional assays. Evaluation of this transcript in normal and leukemia cells suggests that BRCA2(Δ105) may contribute to the viability of persons inheriting this mutation. In this study, we have also characterized 5 other BRCA2 variants and found 3 of these (p.L2510P, p.R2336H, and p.W2626C) to be deleterious and 2 (p.I2490T and p.K2729N) probably neutral. Such studies are important to understand the functional significance of unclassified BRCA2 variants.

Partially Unfolded Forms of the Prion Protein Populated under Misfolding-promoting Conditions CHARACTERIZATION BY HYDROGEN EXCHANGE MASS SPECTROMETRY AND NMR

Background: Folding intermediates of proteins are known to initiate misfolding. Results: Two partially unfolded forms (PUFs) of the prion protein have been characterized structurally and energetically. Conclusion: One of the PUFs is structurally similar to an initial intermediate in prion misfolding. Significance: Identification of aggregation-prone intermediates on the prion proteins folding pathway is the key to understanding its amyloidogenic propensity. The susceptibility of the cellular prion protein (PrPC) to convert to an alternative misfolded conformation (PrPSc), which is the key event in the pathogenesis of prion diseases, is indicative of a conformationally flexible native (N) state. In the present study, hydrogen-deuterium exchange (HDX) in conjunction with mass spectrometry and nuclear magnetic resonance spectroscopy were used for the structural and energetic characterization of the N state of the full-length mouse prion protein, moPrP(23–231), under conditions that favor misfolding. The kinetics of HDX of 34 backbone amide hydrogens in the N state were determined at pH 4. In contrast to the results of previous HDX studies on the human and Syrian hamster prion proteins at a higher pH, various segments of moPrP were found to undergo different extents of subglobal unfolding events at pH 4, a pH at which the protein is known to be primed to misfold to a β-rich conformation. No residual structure around the disulfide bond was observed for the unfolded state at pH 4. The N state of the prion protein was observed to be at equilibrium with at least two partially unfolded forms (PUFs). These PUFs, which are accessed by stochastic fluctuations of the N state, have altered surface area exposure relative to the N state. One of these PUFs resembles a conformation previously implicated to be an initial intermediate in the conversion of monomeric protein into misfolded oligomer at pH 4.

Structural Biophysics of the NusB:NusE Antitermination Complex

In prokaryotic transcription regulation, several host factors form a complex with RNA polymerase and the nascent mRNA. As part of a process known as antitermination, two of these host factors, NusB and NusE, bind to form a heterodimer, which interacts with a specific boxA site on the RNA. The NusB/NusE/boxA RNA ternary complex interacts with the RNA polymerase transcription complex, stabilizing it and allowing transcription past premature termination points. The NusB protein also binds boxA RNA individually and retains all specificity for boxA. However, NusE increases the affinity of RNA to NusB in the ternary complex, which contributes to efficient antitermination. To understand the molecular mechanism of the process, we have determined the structure of NusB from the thermophilic bacterium Aquifex aeolicus and studied the interaction of NusB and NusE. We characterize this binding interaction using NMR, isothermal titration calorimetry, gel filtration, and analytical ultracentrifugation. The binding site of NusE on NusB was determined using NMR chemical shift perturbation studies. We have also determined the NusE binding site in the ternary Escherichia coli NusB/NusE/boxA RNA complex and show that it is very similar to that in the NusB/NusE complex. There is one loop of residues (from 113 to 118 in NusB) affected by NusE binding in the ternary complex but not in the binary complex. This difference may be correlated to an increase in binding affinity of RNA for the NusB/NusE complex.

Salt-Mediated Oligomerization of the Mouse Prion Protein Monitored by Real-Time NMR

The prion protein forms β-rich soluble oligomers in vitro at pH4 in the presence of physiological concentrations of salt. In the absence of salt, oligomerization and misfolding do not take place in an experimentally tractable timescale. While it is well established that a lowering of pH facilitates misfolding and oligomerization of this protein, the role of salt remains poorly understood. Here, solution-state NMR was used to probe perturbations in the monomeric mouse prion protein structure immediately upon salt addition, prior to the commencement of the oligomerization reaction. The weak binding of salt at multiple sites dispersed all over the monomeric protein causes a weak and non-specific perturbation of structure throughout the protein. The only significant perturbation occurs in the loop between helix 2 and 3 in and around the partially buried K193-E195 salt bridge. The disruption of this key electrostatic interaction is the earliest detectable change in the monomer before any major conformational change occurs and appears to constitute the trigger for the commencement of misfolding and oligomerization. Subsequently, the kinetics of monomer loss, due to oligomerization, was monitored at the individual residue level. The oligomerization reaction was found to be rate-limited by association and not conformational change, with an average reaction order of 2.6 across residues. Not surprisingly, salt accelerated the oligomerization kinetics, in a non-specific manner, by electrostatic screening of the highly charged monomers at acidic pH. Together, these results allowed a demarcation of the specific and non-specific effects of salt on prion protein misfolding and oligomerization.

Indomethacin Elicits Proteasomal Dysfunctions Develops Apoptosis Through Mitochondrial Abnormalities

Non‐steroidal anti‐inflammatory drugs (NSAIDs) are a class of drugs that are mainly used to treat pain, inflammation, and fever via cyclooxygenase‐2 (COX‐2) inhibition. There are abundant findings that uncover the hidden critical chemotherapeutics potential of NSAIDs in cancer treatment. However, still the precise mechanism by which NSAIDs could be used as an effective anti‐tumor agent in the prevention of carcinogenesis is not well understood. Here, we show that indomethacin, a well‐known NSAID, induces proteasomal dysfunction that results in accumulation of unwanted proteins, mitochondrial abnormalities, and successively stimulate apoptosis in cells. We observed the interaction of indomethacin with proteasome and noticed the massive accumulation of intracellular ubiquitin‐positive proteins, which might be due to the suppression of proteasome activities. Furthermore, we also found that exposure of indomethacin causes the accumulation of critical proteasomal substrates that consequently generate severe mitochondrial abnormalities and prompt up key apoptotic events in cells. Our results demonstrate how indomethacin affects normal proteasomal functions and induces mitochondrial apoptosis in cells. These findings also improve our current understanding of how NSAIDs can exhibit crucial anti‐proliferative effects in cells. In near future, our findings may suggest a new possible strategy for the development of specific proteasome inhibitors in conjunction with other chemo‐preventive anticancer agents.

Amino-acid composition after loop deletion drives domain swapping

Rational engineering of a protein to enable domain swapping requires an understanding of the sequence, structural and energetic factors that favor the domain‐swapped oligomer over the monomer. While it is known that the deletion of loops between β‐strands can promote domain swapping, the spliced sequence at the position of the loop deletion is thought to have a minimal role to play in such domain swapping. Here, two loop‐deletion mutants of the non‐domain‐swapping protein monellin, frame‐shifted by a single residue, were designed. Although the spliced sequence in the two mutants differed by only one residue at the site of the deletion, only one of them (YEIKG) promoted domain swapping. The mutant containing the spliced sequence YENKG was entirely monomeric. This new understanding that the domain swapping propensity after loop deletion may depend critically on the chemical composition of the shortened loop will facilitate the rational design of domain swapping.

Structural and functional analysis of SMO-1, the SUMO homolog in Caenorhabditis elegans

SUMO proteins are important post-translational modifiers involved in multiple cellular pathways in eukaryotes, especially during the different developmental stages in multicellular organisms. The nematode C. elegans is a well known model system for studying metazoan development and has a single SUMO homolog, SMO-1. Interestingly, SMO-1 modification is linked to embryogenesis and development in the nematode. However, high-resolution information about SMO-1 and the mechanism of its conjugation is lacking. In this work, we report the high-resolution three dimensional structure of SMO-1 solved by NMR spectroscopy. SMO-1 has flexible N-terminal and C-terminal tails on either side of a rigid beta-grasp folded core. While the sequence of SMO-1 is more similar to SUMO1, the electrostatic surface features of SMO-1 resemble more with SUMO2/3. SMO-1 can bind to typical SUMO Interacting Motifs (SIMs). SMO-1 can also conjugate to a typical SUMOylation consensus site as well as to its natural substrate HMR-1. Poly-SMO-1 chains were observed in-vitro even though SMO-1 lacks any consensus SUMOylation site. Typical deSUMOylation enzymes like Senp2 can cleave the poly-SMO-1 chains. Despite being a single gene, the SMO-1 structure allows it to function in a large repertoire of signaling pathways involving SUMO in C. elegans. Structural and functional features of SMO-1 studies described here will be useful to understand its role in development.