Structural Biology Select，2010年2月
6 02 2010Available online 5 February 2010.
Derived from the Greek roots allos and stereos, “allostery” literally means “other object.” Indeed, allosteric mechanisms are often overshadowed by the main action at the active site of an enzyme. This Structural Biology Select summarizes recent studies that highlight allostery’s central role in fundamental cellular processes and in the development of new drugs.
| 免费文献下载 生物医药招聘 检索免费文献 免费英文文献 |
Leukemia Gets the Knockout Punch
The tyrosine kinase inhibitor Gleevec (imatinib) has revolutionized the treatment of chronic myeloid leukemia (CML). CML is caused by a 9:22 chromosomal rearrangement that results in the production of a constitutively active kinase, the BCR-ABL protein, which is the target of Gleevec. Patients in advanced stages of CML often relapse, despite Gleevec treatment, because of the emergence of drug-resistant mutations in the active site of BCR-ABL. Now, Zhang et al. (2010) present comprehensive in vitro and in vivo data showing that allosteric regulators of Bcr-Abl work synergistically with current therapeutics to stop an untreatable form of Bcr-Abl that contains a T315I mutation. The study focuses on a new class of inhibitors, known as GNFs. When mixed with standard kinase inhibitors, such as Gleevec, GNF compounds cooperatively block enzymatic activity of the T315I mutant Bcr-Abl and halt growth of a cultured cell line derived from mouse bone marrow that expresses this fusion protein. In addition, this tag-team therapy decreases the emergence of resistant clones by 90% and, most importantly, results in complete disease remission in a T315I Bcr-Abl mouse model. X-ray crystal structures and hydrogen-deuterium exchange experiments show that, unlike Gleevec (which interacts directly with the active site of Bcr-Abl), GNF inhibitors insert themselves into the myristate-binding pocket of the Bcr-Abl kinase. This allosterically perturbs the dynamics of the active site and provides the coup de grâce for stabilizing the off-state of T315I Bcr-Abl protein. Receptor tyrosine kinases, such as the epidermal growth factor receptor and the platelet-derived growth factor receptor-β, also develop drug-resistant mutations that are similar to T315I in BCR-ABL. Therefore, allosteric inhibitors directed at these receptors may also prove useful for treating other cancers, including those of lung, breast, and pancreas.
Zhang et al. (2010). Nature. Published online January 13, 2010. 10.1038/nature08675.
A helicopter turns off the Bcr-Abl kinase (pink) by airlifting the GNF-2 inhibitor (balls and sticks) into the myristate-binding pocket of Bcr-Abl (yellow). Figure courtesy of Nathanael S. Gray and Jianming Zhang.
The extracellular surface of the β2-adrenergic receptor (blue) embedded in a phospholipid bilayer (gray and red spheres). Figure courtesy of Xavier Deupi.
The MID domain of the Argonaute protein (Ago1) interacts allosterically with an miRNA (red) and the 5′ cap of the target mRNA (purple). Figure courtesy of Michelle Kim Zinchenko.
Rho and RNAP Finally Tie the Knot
Could allostery also be critical for Rho-dependent transcription termination in bacteria? The RNA helicase Rho uses the energy of ATP hydrolysis to move along an emerging nascent strand of RNA. When Rho reaches an actively transcribing RNA polymerase (RNAP), it disrupts elongation, and the RNAP molecule falls off the template DNA. Recent X-ray structures of Rho in a complex with RNA provide stunning details of Rho’s translocation mechanism, but how Rho forces RNAP to dissociate from DNA and RNA is still unknown. Now, Epshtein et al. (2010) present compelling evidence that RNAP plays an active role in Rho-dependent termination. In vitro transcription assays indicate that Rho binds to the RNAP protein complex in the absence of DNA (or RNA) and in the presence of extremely short RNAs, suggesting that Rho and RNAP interact not only at termination but possibly throughout the transcription cycle. Mutations in the lid domain of RNAP strongly hindered Rho-induced termination, and RNA crosslinking studies identified a Rho-dependent conformational change in the catalytic core of RNAP, specifically at the β′-trigger loop. From these results, the authors propose a termination mechanism in which Rho allosterically drives a structural rearrangement at the β′-trigger loop of RNAP by “pushing” on RNAP’s lid domain. They hypothesize that the remodeling of the catalytic core triggers the opening of the RNAP elongation complex, resulting in RNAP dissociation from RNA and DNA. Although future studies with complementary techniques are required to confirm and refine the details of this model, these new data illuminate the complex allosteric mechanisms that arise when a powerful ATPase, such as Rho, comes into contact with a sophisticated molecular machine, such as RNAP.
Epshtein et al. (2010). Nature 463, 245–249.
Michaeleen Doucleff
Locking Down TetR Regulation
A better understanding of the allosteric regulation of the tetracycline repressor (TetR) in bacteria may also uncover new routes for improving antibiotics. When tetracycline interacts with TetR’s N-terminal domain, a conformational change 30 Å away in the C-terminal DNA-binding domain releases the repressor from promoter DNA and induces genes required for clearance of the drug. Previous X-ray crystal structures suggested that the regulation of TetR by tetracycline involved a simple switch between two static configurations with different affinities for DNA. Now, a study by Reichheld et al. (2009) challenges this classic view of allostery. The authors propose that tetracycline’s interaction at the N-terminal domain decreases the conformational space sampled by the C-terminal domain and “locks” it into a configuration that is incompatible with DNA recognition. By carefully examining denaturation curves, the authors find that tetracycline significantly increases the thermodynamic stability of TetR’s DNA-binding domain. In addition, the affinity of different TetR mutants for DNA correlates with the inherent flexibility of the mutant’s DNA-binding domain. A re-examination of X-ray crystal structures suggests that the long-distance regulation of thermodynamics is probably propagated through TetR’s hydrophobic core. This new model, which links the repressor’s activity to its thermodynamic stability, provides a unified explanation for the complex behavior that was previously observed for TetR mutant phenotypes, and it offers a possible mechanism for how this highly conserved family of repressors responds to chemically diverse molecules. It will be exciting to see how well this model applies to the other 2353 TetR family members and to bacterial repressors and activators in general.
Reichheld et al. (2009). Proc. Natl. Acad. Sci. USA 106, 22263–22268.
Argonaute CAPtivates miRNA
A more classical type of allosteric regulation was recently uncovered in the Argonaute protein family. MicroRNAs (miRNAs) unite with an Argonaute protein to silence messenger RNA (mRNA) transcripts possessing imperfect complementarity to the miRNA. However, the pathway that guides the identification of the target mRNAs is still poorly understood. Using a suite of bioinformatic and biochemical approaches, Djuranovic et al. (2010) recently discovered a pair of allosteric binding sites in the Argonaute MID domain that may help the RNA-silencing complex to trigger translational repression of the correct mRNA targets at the appropriate time. Clustering analysis of the MID domain sequences for the entire Argonaute family separated the proteins that associate with miRNAs from other classes of Argonaute proteins, including those that associate with small interfering RNAs (siRNAs). This functional-based grouping of the MID domains strongly implicates this region as a key differentiator between the siRNA and miRNA silencing pathways. And, indeed, the authors found major differences in the nucleic acid binding behavior of the two classes of Argonaute proteins. For factors that associate with miRNAs, the presence of triphosphate nucleotides (compounds that mimic the 5′ cap) increases their binding affinity for miRNAs; and, similarly, the presence of miRNAs increases the binding affinity of these Argonaute proteins for 5′-capped mRNAs. In contrast, this allostery could not be detected for Argonaute proteins that associate with siRNAs. The authors then used an in vivo reporter gene assay and mutagenesis to pinpoint the location of a putative 5′ cap-binding site on the Drosophila Argonaute-1 protein (DmAgo1) and to demonstrate that the association of DmAgo1 with both a 5′ cap and an miRNA correlates with translational repression. This study raises many fresh questions about silencing by miRNA-Argonaute complexes. Is the allosteric regulation evoked during the initial loading of miRNA or during silencing itself? How do the allosteric structural changes affect the other domains in the Argonaute protein? And how well are these allosteric sites conserved across eukaryotes?
Djuranovic et al. (2010). Nat. Struct. Mol. Biol. Published online January 10, 2010. 10.1038/nsmb.1736.
Scratching the Surface of GPCR Regulators
In addition to thwarting drug-resistant CML, allostery may also be key to developing “smart” drugs that can discriminate among closely related G protein-coupled receptors (GPCRs). GPCRs are the target for about half of all modern pharmaceuticals, but the production of agents that are selective for specific GPCR subtypes has been hampered by the remarkable structural similarity of their drug binding pockets. In contrast, the extracellular surfaces of GPCRs possess surprising diversity, even between receptor subtypes, such as the β1- and β2-adrenergic receptors. Using a novel nuclear magnetic resonance (NMR) spectroscopy method for probing the structural dynamics of GPCRs, Bokoch et al. (2010) have identified a motif on the extracellular surface of the β2-adrenergic receptor that directly reports on the activation state of the cytoplasmic surface, making this region an ideal target for highly selective GPCR modulators. GPCRs are generally off limits for traditional NMR studies because of the low signal-to-noise ratio generated by a receptor embedded in lipids. Bokoch et al. (2010) overcome this roadblock by developing a novel chemical trick for labeling lysine residues with 13C-methyl groups, which possess a high signal-to-noise ratio and allow easy monitoring of lysine residues on the surface of a protein. With this tool and computational modeling, the authors show that extracellular loops 2 and 3 of the β2-adrenergic receptor exist in three distinct configurations depending on the type of ligand bound to the transmembrane core (neutral antagonists, agonists, or inverse agonists). Therefore, this region provides a direct readout of the GPCR’s activation level. The authors argue that small molecules stabilizing one of these three configurations of the GPCR may be able to regulate allosterically the strength of its coupling to G proteins. This study introduces an exciting biophysical tool for characterizing GPCR dynamics that cannot be detected by static X-ray structures. It also reveals a new route for designing allosteric modulators for the β2-adrenergic receptor with seemingly laser-guided selectivity.
Bokoch et al. (2010). Nature 463, 108–112.
