Different Poses for Ligand and Chaperone in Inhibitor-Bound Hsp90 and GRP94: Implications for Paralog-Specific Drug Design

Hsp90 chaperones contain an N-terminal ATP binding site that has been effectively targeted by competitive inhibitors. Despite the myriad of inhibitors, none to date have been designed to bind specifically to just one of the four mammalian Hsp90 paralogs, which are cytoplasmic Hsp90α and β, endoplasmic reticulum GRP94, and mitochondrial Trap-1. Given that each of the Hsp90 paralogs is responsible for chaperoning a distinct set of client proteins, specific targeting of one Hsp90 paralog may result in higher efficacy and therapeutic control. Specific inhibitors may also help elucidate the biochemical roles of each Hsp90 paralog. Here, we present side-by-side comparisons of the structures of yeast Hsp90 and mammalian GRP94, bound to the pan-Hsp90 inhibitors geldanamycin (Gdm) and radamide. These structures reveal paralog-specific differences in the Hsp90 and GRP94 conformations in response to Gdm binding. We also report significant variation in the pose and disparate binding affinities for the Gdm-radicicol chimera radamide when bound to the two paralogs, which may be exploited in the design of paralog-specific inhibitors.     

Hydration Structures of the Human Protein Kinase CK2α Clarified by Joint Neutron and X-ray Crystallography

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Å resolution) and X-ray (1.1 Å resolution) crystallography. The analysis revealed the structure of conserved water molecules at the active site and a long potential hydrogen bonding network originating from the catalytic Asp156 that is well known to enhance the nucleophilicity of the substrate OH group to the γ-phospho group of ATP by proton elimination. His148 and Asp214 conserved in the protein kinase family are located in the middle of the network. The water molecule forming a hydrogen bond with Asp214 appears to be deformed. In addition, mutational analysis of His148 in CK2 showed significant reductions by 40%–75% in the catalytic efficiency with similar affinity for ATP. Likewise, remarkable reductions to less than 5% were shown by corresponding mutations on His131 in death-associated protein kinase 1, which belongs to a group different from that of CK2. These findings shed new light on the catalytic mechanism of protein kinases in which the hydrogen bond network through the C-terminal domain may assist the general base catalyst to extract a proton with a link to the bulk solvent via intermediates of a pair of residues.     

Structural Basis for Bivalent Smac-Mimetics Recognition in the IAP Protein Family

XIAP is an apoptotic regulator protein that binds to the effector caspases -3 and -7 through its BIR2 domain, and to initiator caspase-9 through its BIR3 domain. Molecular docking studies suggested that Smac-DIABLO may antagonize XIAP by concurrently targeting both BIR2 and BIR3 domains; on this basis bivalent Smac-mimetic compounds have been proposed and characterized. Here, we report the X-ray crystal structure of XIAP-BIR3 domain in complex with a two-headed compound (compound 3) with improved efficacy relative to its monomeric form. A small-angle X-ray scattering study of XIAP-BIR2BIR3, together with fluorescence polarization binding assays and compound 3 cytotoxicity tests on HL60 leukemia cell line are also reported. The crystal structure analysis reveals a network of interactions supporting XIAP-BIR3/compound 3 recognition; moreover, analytical gel-filtration chromatography shows that compound 3 forms a 1:1 stoichiometric complex with a XIAP protein construct containing both BIR2 and BIR3 domains. On the basis of the crystal structure and small-angle X-ray scattering, a model of the same BIR2-BIR3 construct bound to compound 3 is proposed, shedding light on the ability of compound 3 to relieve XIAP inhibitory effects on caspase-9 as well as caspases -3 and -7. A molecular modeling/docking analysis of compound 3 bound to cIAP1-BIR3 domain is presented, considering that Smac-mimetics have been shown to kill tumor cells by inducing cIAP1 and cIAP2 ubiquitination and degradation. Taken together, the results reported here provide a rationale for further development of compound 3 as a lead in the design of dimeric Smac mimetics for cancer treatment.     

Structural and computational investigations of VIM-7: insights into the substrate specificity of vim metallo-β-lactamases

The presence of metallo-β-lactamases (MBLs) in many clinically important human bacterial pathogens limits treatment options, as these enzymes efficiently hydrolyze nearly all β-lactam antibiotics. VIM enzymes are among the most widely distributed MBLs, but many of the individual VIM subtypes remain poorly characterized. Pseudomonas aeruginosa VIM-7 is the most divergent among VIM-type MBLs in terms of amino acid sequence. Here we present crystal structures of VIM-7 as the native enzyme, with Cys221 oxidized (VIM-7-Ox), and with a sulfur atom bridging the two active-site zinc ions (VIM-7-S). Comparison with VIM-2 and VIM-4 structures suggests an explanation for the reduced catalytic efficiency of VIM-7 against cephalosporins with a positively charged cyclic substituent at the C3 position (e.g., ceftazidime). Kinetic variations are attributed to substitutions in residues 60-66 (that form a loop adjacent to the active site previously implicated in substrate binding) and to the disruption of two hydrogen-bonding clusters through substitutions at positions 218 and 224. Furthermore, the less negatively charged surface of VIM-7 (compared to VIM-2) may also contribute to the reduced hydrolytic efficiency. Docking of the cephalosporins ceftazidime and cefotaxime into the VIM-2 and VIM-7 structures reveals that amino acid substitutions may cause the mode of substrate binding to differ between the two enzymes. Our structures thus provide new insights into the variation in substrate specificity that is evident across this family of clinically important enzymes.     

Molecular determinants of substrate specificity in plant 5'-methylthioadenosine nucleosidases

5′-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5′-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5′-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 Å resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5′-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5′-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pK_a of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN.     

Large Power Amplification in Magneto‐Mechano‐Electric Harvesters through Distributed Forcing

Energy harvesting from extremely low frequency magnetic fields using magneto‐mechano‐electric (MME) harvesters enables wireless power transfer for operating Internet of Things (IoT) devices. The MME harvesters are designed to resonate at a fixed frequency by absorbing AC magnetic fields through a composite cantilever comprising of piezoelectric and magnetostrictive materials, and a permanent magnetic tip mass. However, this harvester architecture limits power generation because volume of the magnetic end mass is closely coupled with the resonance frequency of the device structure. Here, a method is demonstrated for maintaining the resonance frequency of the MME harvesters under all operating conditions (e.g., 60 Hz, standard frequency of electricity in many countries) while simultaneously enhancing the output power generation. By distributing the magnetic mass over the beam, the output power of the harvester is significantly enhanced at a constant resonance frequency. The MME harvester with distributed forcing shows 280% improvement in the power generation compared with a traditional architecture. The generated power is shown to be sufficient to power eight different onboard sensors with wireless data transmission integrated on a drone. These results demonstrate the promise of MME energy harvesters for powering wireless communication and IoT sensors.     

Structural insights into rice BGlu1 beta-glucosidase oligosaccharide hydrolysis and transglycosylation

The structures of rice BGlu1 β-glucosidase, a plant β-glucosidase active in hydrolyzing cell wall-derived oligosaccharides, and its covalent intermediate with 2-deoxy-2-fluoroglucoside have been solved at 2.2 Å and 1.55 Å resolution, respectively. The structures were similar to the known structures of other glycosyl hydrolase family 1 (GH1) β-glucosidases, but showed several differences in the loops around the active site, which lead to an open active site with a narrow slot at the bottom, compatible with the hydrolysis of long β-1,4-linked oligosaccharides. Though this active site structure is somewhat similar to that of the Paenibacillus polymyxa β-glucosidase B, which hydrolyzes similar oligosaccharides, molecular docking studies indicate that the residues interacting with the substrate beyond the conserved -1 site are completely different, reflecting the independent evolution of plant and microbial GH1 exo-β-glucanase/β-glucosidases. The complex with the 2-fluoroglucoside included a glycerol molecule, which appears to be in a position to make a nucleophilic attack on the anomeric carbon in a transglycosylation reaction. The coordination of the hydroxyl groups suggests that sugars are positioned as acceptors for transglycosylation by their interactions with E176, the catalytic acid/base, and Y131, which is conserved in barley BGQ60/β-II β-glucosidase, that has oligosaccharide hydrolysis and transglycosylation activity similar to rice BGlu1. As the rice and barley enzymes have different preferences for cellobiose and cellotriose, residues that appeared to interact with docked oligosaccharides were mutated to those of the barley enzyme to see if the relative activities of rice BGlu1 toward these substrates could be changed to those of BGQ60. Although no single residue appeared to be responsible for these differences, I179, N190 and N245 did appear to interact with the substrates.     

Symmetry and asymmetry of the RING-RING dimer of Rad18

The human ubiquitin-conjugating enzyme Rad6 (E2), with ubiquitin ligase enzyme Rad18 (RING E3), monoubiquitinates proliferating cell nuclear antigen at stalled replication forks in DNA translesion synthesis. Here, we determine the structure of the homodimeric Rad18 RING domains by X-ray crystallography and classify it to RING-RING dimers that dimerize through helices adjacent to the RING domains and through the canonical RING domains. Using NMR spectroscopy and site-directed mutagenesis, we demonstrate that the Rad6b binding site, for the Rad18 RING domain, strongly resembles that of other E2/E3 RING/U-box complexes. We show that the homodimeric Rad18 RING domain can recruit two Rad6b E2 enzymes, whereas the full-length Rad18 homodimer binds only to a single Rad6b molecule. Such asymmetry is a common feature of RING-RING heterodimers and has been observed for the CHIP U-box homodimer. We propose that asymmetry may be a common feature of dimeric RING E3 ligases.     

Coevolution of Antibody Stability and Vκ CDR-L3 Canonical Structure

Antibodies recognize antigens through six hypervariable loops, five of which have a limited set of conformations known as canonical structures. For κ light chains, the majority of CDR-L3 [the third hypervariable loop of the light chain variable domain (V_L)] adopts the type 1 canonical structure (CS1), with a cis-proline at position 95. Here, we present the design and structural studies of the monoclonal antibody mAb15 and related mutants that contained a series of progressively germline mutations only in the heavy chain variable domain (V_H) that ultimately led to an increase of more than 11 °C in the melting temperature (T_m) of the antigen-binding fragment (Fab). The all-trans CDR-L3 structure in the wild type is significantly different from any known CDR-L3 canonical structures. In the thermally stable mutants, the L94^L-S95^L peptide bond adopts an energetically unfavorable non-X-proline cis conformation, but the overall CDR-L3 loop converted to CS1. The stabilized V_H appears to function as a specific molecular chaperone that facilitated the trans-cis isomerization of S95^L. Thus, it is plausible that proline is the evolutionary choice to maintain overall structure and stability for V_L. These results provide new insights into the evolution of CS1 and suggest a potential molecular switch mechanism at position 95 that links CDR-L3 structural diversity and antibody stability and will have implications for antibody engineering.     

Simulating RNA folding kinetics on approximated energy landscapes

We present a general computational approach to simulate RNA folding kinetics that can be used to extract population kinetics, folding rates and the formation of particular substructures that might be intermediates in the folding process. Simulating RNA folding kinetics can provide unique insight into RNA whose functions are dictated by folding kinetics and not always by nucleotide sequence or the structure of the lowest free-energy state. The method first builds an approximate map (or model) of the folding energy landscape from which the population kinetics are analyzed by solving the master equation on the map. We present results obtained using an analysis technique, map-based Monte Carlo simulation, which stochastically extracts folding pathways from the map. Our method compares favorably with other computational methods that begin with a comprehensive free-energy landscape, illustrating that the smaller, approximate map captures the major features of the complete energy landscape. As a result, our method scales to larger RNAs. For example, here we validate kinetics of RNA of more than 200 nucleotides. Our method accurately computes the kinetics-based functional rates of wild-type and mutant ColE1 RNAII and MS2 phage RNAs showing excellent agreement with experiment.