Sujoy Mukherjee

Lipophilic bisphosphonates as dual farnesyl/geranylgeranyl diphosphate synthase inhibitors: an X-ray and NMR investigation.

Considerable effort has focused on the development of selective protein farnesyl transferase (FTase) and protein geranylgeranyl transferase (GGTase) inhibitors as cancer chemotherapeutics. Here, we report a new strategy for anticancer therapeutic agents involving inhibition of farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), the two enzymes upstream of FTase and GGTase, by lipophilic bisphosphonates. Due to dual site targeting and decreased polarity, the compounds have activities far greater than do current bisphosphonate drugs in inhibiting tumor cell growth and invasiveness, both in vitro and in vivo. We explore how these compounds inhibit cell growth and how cell activity can be predicted based on enzyme inhibition data, and using X-ray diffraction, solid state NMR, and isothermal titration calorimetry, we show how these compounds bind to FPPS and/or GGPPS.

Thermodynamics of Bisphosphonates Binding to Human Bone: A Two-Site Model

We have used isothermal titration calorimetry (ITC) to study the thermodynamics of binding of 12 bisphosphonates to human bone. The ITC results show that there are two binding sites. Site A is the weak, highly populated site seen by NMR and is characterized by an average DeltaG of binding of -5.2 kcal. Site B is a strong binding site characterized by a DeltaG of binding of -8.5 kcal. Binding to both sites is overwhelmingly entropy driven. Using a thermodynamic group approach and a linear regression method, we predict the DeltaG of binding of all 12 compounds with an R(2) = 0.95 (a 0.19 kcal error variance estimate, approximately 3% of the total DeltaG range), opening up the way to designing novel chemotherapy, immunotherapy, and anti-infectious disease drugs having weak bone binding affinity.

NMR Investigations of the Static and Dynamic Structures of Bisphosphonates on Human Bone: a Molecular Model

We report the results of an investigation of the binding of a series of bisphosphonate drugs to human bone using 2H, 13C, 15N, and 31P nuclear magnetic resonance spectroscopy. The 31P NMR results show that the bisphosphonate groups bind irrotationally to bone, displacing orthophosphate from the bone mineral matrix. Binding of pamidronate is well described by a Langmuir-like isotherm, from which we deduce an approximately 30-38 A2 surface area per pamidronate molecule and a deltaG = -4.3 kcal mol(-1). TEDOR of [13C3, 15N] pamidronate on bone shows that the bisphosphonate binds in a gauche [N-C(1)] conformation. The results of 31P as well as 15N shift and cross-polarization measurements indicate that risedronate binds weakly, since it has a primarily neutral pyridine side chain, whereas zoledronate (with an imidazole ring) binds more strongly, since the ring is partially protonated. The results of 2H NMR measurements of side-chain 2H-labeled pamidronate, alendronate, zoledronate, and risedronate on bone show that all side chains undergo fast but restricted motions. In pamidronate, the motion is well simulated by a gauche+/gauche- hopping motion of the terminal -CH2-NH3(+) group, due to jumps from one anionic surface group to another. The results of double-cross polarization experiments indicate that the NH3(+)-terminus of pamidronate is close to the bone mineral surface, and a detailed model is proposed in which the gauche side-chain hops between two bone PO4(3-) sites.

Activator Gcn4 Employs Multiple Segments of Med15/Gal11, Including the KIX Domain, to Recruit Mediator to Target Genes in Vivo

Mediator is a multisubunit coactivator required for initiation by RNA polymerase II. The Mediator tail subdomain, containing Med15/Gal11, is a target of the activator Gcn4 in vivo, critical for recruitment of native Mediator or the Mediator tail subdomain present in sin4Δ cells. Although several Gal11 segments were previously shown to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We show that interaction of Gcn4 with the Mediator tail in vitro and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo involve additive contributions from three different segments in the N terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we establish that B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrate that Gcn4 binds to the Gal11 KIX domain directly and, by NMR chemical shift analysis combined with mutational studies, we identify the likely binding site for Gcn4 on the KIX surface. Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator in vivo.

Conformational flexibility of a human immunoglobulin light chain variable domain by relaxation dispersion nuclear magnetic resonance spectroscopy: implications for protein misfolding and amyloid assembly.

The conformational flexibility of a human immunoglobulin κIV light-chain variable domain, LEN, which can undergo conversion to amyloid under destabilizing conditions, was investigated at physiological and acidic pH on a residue-specific basis by multidimensional solution-state nuclear magnetic resonance (NMR) methods. Measurements of backbone chemical shifts and amide (15)N longitudinal and transverse spin relaxation rates and steady-state nuclear Overhauser enhancements indicate that, on the whole, LEN retains its native three-dimensional fold and dimeric state at pH 2 and that the protein backbone exhibits limited fast motions on the picosecond to nanosecond time scale. On the other hand, (15)N Carr--Purcell--Meiboom--Gill (CPMG) relaxation dispersion NMR data show that LEN experiences considerable slower, millisecond time scale dynamics, confined primarily to three contiguous segments of about 5-20 residues and encompassing the N-terminal β-strand and complementarity determining loop regions 2 and 3 in the vicinity of the dimer interface. Quantitative analysis of the CPMG relaxation dispersion data reveals that at physiological pH these slow backbone motions are associated with relatively low excited-state protein conformer populations, in the ~2-4% range. Upon acidification, the minor conformer populations increase significantly, to ~10-15%, with most residues involved in stabilizing interactions across the dimer interface displaying increased flexibility. These findings provide molecular-level insights about partial protein unfolding at low pH and point to the LEN dimer dissociation, initiated by increased conformational flexibility in several well-defined regions, as being one of the important early events leading to amyloid assembly.

Conformational Flexibility Tunes the Propensity of Transthyretin to Form Fibrils Through Non‐Native Intermediate States

The formation of partially unfolded intermediates through conformational excursions out of the native state is the starting point of many diseases involving protein aggregation. Therapeutic strategies often aim to stabilize the native structure and prevent the formation of intermediates that are also cytotoxic in vivo. However, their transient nature and low population makes it difficult to characterize these intermediates. We have probed the backbone dynamics of transthyretin (TTR) over an extended timescale by using NMR spectroscopy and MD simulations. The location and extent of these motions indicates that the backbone flexibility of TTR is a cause of dissociation and destabilization, both of which are responsible for fibril formation. Importantly, approximately 10 % of wild-type TTR exists in an intermediate state, which increased to up to 28 % for pathogenic TTR mutants, for which the formation of the intermediate state is shown to be energetically more favorable compared to the wild type. This result suggests an important role for the intermediates in TTR amyloidosis.

Backbone and side-chain 1H, 13C and 15N resonance assignments of LEN, a human immunoglobulin κIV light-chain variable domain

Abstract1H, 13C and 15N resonance assignments are presented for a recombinant 114 amino acid human immunoglobulin (Ig) κIV light-chain variable domain (VL) LEN, which displays a high degree of sequence identity with another human Ig κIV VL, SMA. While SMA is highly amyloidogenic in vivo and in vitro and has been linked to the pathogenesis of light-chain amyloidosis, LEN is non-amyloidogenic in vivo and can be converted to the amyloid state only in vitro under destabilizing conditions. Measurements of longitudinal and transverse amide 15N relaxation rates confirm that, as expected, LEN is a dimer at physiological pH and typical concentrations used for NMR studies, and the analysis of secondary chemical shifts indicates that the protein has a high β-sheet content. These findings are consistent with previously published biophysical data and the high-resolution X-ray structure of LEN.

Skeletal Muscle Dystrophy mutant of lamin A alters the structure and dynamics of the Ig fold domain

Mutations in the different domains of A-type lamin proteins cause a diverse plethora of diseases collectively termed as laminopathies which can affect multiple organs. Ig fold is one such domain of lamin A which is implicated in numerous nuclear interactions wherein the mutations lead to different laminopathies. W514R is one such mutation in the Ig fold which leads to severe phenotypes in Skeletal Muscle Dystrophy (SMD) which is a class of laminopathies. In this report, we elucidated gross alterations in structure and dynamics at the level of individual amino acids. These studies indicate altered conformational features of residues in the close vicinity of W514. Imaging of mammalian cells transfected with the mutant have shown distinct perturbation of the nuclear meshwork with concomitant alteration in nuclear interactions as a result of increased oligomerization of Ig W514R. Hence, this novel approach of amalgamating theoretical and experimental procedures to predict the severity of a mutant in the context of laminopathies could be extended for numerous lamin A mutants.

Cloning, expression, purification and characterization of oligomeric states of the native 5HT2A G-Protein-Coupled Receptor

BACKGROUND The 5HT2A G-Protein Coupled Receptor (GPCR) is an important family of receptors involved in an array of neuromodulatory functions. Their dysregulation has been implicated in a number of psychiatric diseases. In spite of the importance of this GPCR, high resolution structure and mechanistic details of its function is unknown. Cholesterol plays an important role in the function of many receptors and reduced cholesterol levels can lead to disruption of serotonergic pathways. However, the role of cholesterol in the formation of GPCR oligomers has not been previously shown for this receptor. Given that receptor dimers have been shown to be the functional unit of this receptor, it is important to investigate the effect of cholesterol in the oligomeric state of 5HT2A receptor. OBJECTIVES The main objective of this work is to clone, over-express and purify the 5HT2A receptor and investigate the effect of cholesterol in its oligomer formation. METHODS The 5HT2A receptor (5HT2AR) DNA construct was subcloned into pFastBac-HT vector and the purified bacmid was used to transfect healthy Sf9 cells. After subsequent passages, a high titer baculovirus was used for over-expression in Sf9 cells. To verify whether the over-expressed receptor was localized in the membrane or cytosolic fraction, cells with and without baculoviral infection were analyzed by immunocytochemistry. Subsequently, the over-expression conditions required to obtain sufficient quantity of the receptor was optimized followed by the optimization of the purification conditions. Finally, the culture was scaled up and the receptor was purified by affinity chromatography. The over-expression of the receptor was checked by Western blotting and purity was analyzed by Coomassie stained SDS PAGE. Cryo-electron microscopy experiments were performed on the purified receptor in presence and absence of cholesterol and at multiple concentrations to rule out any concentration dependent effect on the oligomer formation. RESULTS Immunocytochemistry experiments showed prominent nuclear staining; however, bright green staining along the cell membrane was observed only for the infected cells, suggesting appropriate trafficking of majority of the over-expressed receptors to the cell membrane. Results of cryoelectron microscopy show that the receptor with cholesterol had particles that were bigger in size (~11 - 12 nm) compared to the dimension of known GPCR homologs. In contrast, the receptor after removal of cholesterol revealed a uniform distribution of smaller particles (~5 - 6 nm) that is approximately half the size of 5HT2AR particles with cholesterol. Comparing the 2D average views of detergent-encapsulated 5HT2AR particles with the overall dimensions of other 5HT receptor analogs, we show that while a 5HT2AR dimer more closely matches the dimensions of particles with CHS, only a monomer can be fit to particles without CHS. Importantly, even at higher receptor concentration and particle density, the size for 5HT2AR particles without CHS remains the same, suggesting that dimerization is unlikely an effect of concentration. CONCLUSION Our results indicated that 5HT2A receptor primarily forms a dimer in presence of cholesterol whereas it predominantly forms a monomer when cholesterol is removed.

Effect of amino acid mutations on the conformational dynamics of amyloidogenic immunoglobulin light-chains: A combined NMR and in silico study

The conformational dynamics of a pathogenic κ4 human immunoglobulin light-chain variable domain, SMA, associated with AL amyloidosis, were investigated by 15N relaxation dispersion NMR spectroscopy. Compared to a homologous light-chain, LEN, which differs from SMA at eight positions but is non-amyloidogenic in vivo, we find that multiple residues in SMA clustered around the N-terminus and CDR loops experience considerable conformational exchange broadening caused by millisecond timescale protein motions, consistent with a destabilized dimer interface. To evaluate the contribution of each amino acid substitution to shaping the dynamic conformational landscape of SMA, NMR studies were performed for each SMA-like point mutant of LEN followed by in silico analysis for a subset of these proteins. These studies show that a combination of only three mutations located within or directly adjacent to CDR3 loop at the dimer interface, which remarkably include both destabilizing (Q89H and Y96Q) and stabilizing (T94H) mutations, largely accounts for the differences in conformational flexibility between LEN and SMA. Collectively, our studies indicate that a correct combination of stabilizing and destabilizing mutations is key for immunoglobulin light-chains populating unfolded intermediates that result in amyloid formation, and underscore the complex nature of correlations between light-chain conformational flexibility, thermodynamic stability and amyloidogenicity.