Alan Merk

2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor

Pushing the limits of electron microscopy Recent advances in cryo–electron microscopy (cryo-EM) allow structures of large macromolecules to be determined at near-atomic resolution. So far, though, resolutions approaching 2 Å, where features key to drug design are revealed, remain the province of x-ray crystallography. Bartesaghi et al. achieved a resolution of 2.2 Å for a 465-kD ligand-bound protein complex using cryo-EM. The density map is detailed enough to show close to 800 water molecules, magnesium and sodium ions, and precise side-chain conformations. These results bring routine use of cryo-EM in rational drug design a step closer. Science, this issue p. 1147 Advances in electron microscopy allow protein structure determination at resolutions useful in drug discovery. Cryo–electron microscopy (cryo-EM) is rapidly emerging as a powerful tool for protein structure determination at high resolution. Here we report the structure of a complex between Escherichia coli β-galactosidase and the cell-permeant inhibitor phenylethyl β-d-thiogalactopyranoside (PETG), determined by cryo-EM at an average resolution of ~2.2 angstroms (Å). Besides the PETG ligand, we identified densities in the map for ~800 water molecules and for magnesium and sodium ions. Although it is likely that continued advances in detector technology may further enhance resolution, our findings demonstrate that preparation of specimens of adequate quality and intrinsic protein flexibility, rather than imaging or image-processing technologies, now represent the major bottlenecks to routinely achieving resolutions close to 2 Å using single-particle cryo-EM.

2.3 A Resolution Cryo-Em Structure of Human P97 and Mechanism of Allosteric Inhibition

AAA ATPase conformational high jinks The protein p97 is an AAA adenosine triphosphatase (ATPase) that uses energy from ATP hydrolysis to regulate substrates involved in intracellular protein quality control. Its role in this central process makes it a target for cancer chemotherapy. Banerjee et al. used cryo-electron microscopy to determine high-resolution structures for multiple conformational states of this dynamic macromolecular machine. They also determined the structure of the ADP-bound state bound to an inhibitor. The structures give insight into nucleotide-driven structural changes that drive function and show how inhibitor binding prevents these conformational changes Science, this issue p. 871 Cryo–electron microscopy reveals atomic-resolution structures of a protein complex that is a target for cancer drug development. p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo–electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)–bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5′-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.

Structure of β-galactosidase at 3.2-Å resolution obtained by cryo-electron microscopy

Significance Atomic resolution models for proteins and protein complexes are usually obtained using X-ray crystallography or NMR spectroscopy, and in selected instances, by cryo-electron microscopy (cryo-EM) of ordered protein assemblies. The vast majority of high-resolution structures obtained using cryo-EM have been typically restricted to large, well-ordered entities such as helical or icosahedral assemblies or two-dimensional crystals. We show here that emerging methods in single-particle cryo-EM now allow structure determination at near-atomic resolution, even for much smaller protein complexes with low symmetry, by determining the structure of the 465-kDa enzyme β-galactosidase. In addition, by quantitative comparison of density maps obtained at different electron dosages, we demonstrate preferential sensitivity of residues such as Asp and Glu to damage upon irradiation with electrons. We report the solution structure of Escherichia coli β-galactosidase (∼465 kDa), solved at ∼3.2-Å resolution by using single-particle cryo-electron microscopy (cryo-EM). Densities for most side chains, including those of residues in the active site, and a catalytic Mg2+ ion can be discerned in the map obtained by cryo-EM. The atomic model derived from our cryo-EM analysis closely matches the 1.7-Å crystal structure with a global rmsd of ∼0.66 Å. There are significant local differences throughout the protein, with clear evidence for conformational changes resulting from contact zones in the crystal lattice. Inspection of the map reveals that although densities for residues with positively charged and neutral side chains are well resolved, systematically weaker densities are observed for residues with negatively charged side chains. We show that the weaker densities for negatively charged residues arise from their greater sensitivity to radiation damage from electron irradiation as determined by comparison of density maps obtained by using electron doses ranging from 10 to 30 e−/Å2. In summary, we establish that it is feasible to use cryo-EM to determine near-atomic resolution structures of protein complexes (<500 kDa) with low symmetry, and that the residue-specific radiation damage that occurs with increasing electron dose can be monitored by using dose fractionation tools available with direct electron detector technology.

HIV-1 envelope glycoprotein structure

The trimeric envelope glycoprotein of HIV-1, composed of gp120 and gp41 subunits, remains a major target for vaccine development. The structures of the core regions of monomeric gp120 and gp41 have been determined previously by X-ray crystallography. New insights into the structure of trimeric HIV-1 envelope glycoproteins are now coming from cryo-electron tomographic studies of the gp120/gp41 trimer as displayed on intact viruses and from cryo-electron microscopic studies of purified, soluble versions of the ectodomain of the trimer. Here, we review recent developments in these fields as they relate to our understanding of the structure and function of HIV-1 envelope glycoproteins.

Cryo-EM Structures of the Magnesium Channel CorA Reveal Symmetry Break upon Gating.

CorA, the major Mg(2+) uptake system in prokaryotes, is gated by intracellular Mg(2+) (KD ∼ 1-2 mM). X-ray crystallographic studies of CorA show similar conformations under Mg(2+)-bound and Mg(2+)-free conditions, but EPR spectroscopic studies reveal large Mg(2+)-driven quaternary conformational changes. Here, we determined cryo-EM structures of CorA in the Mg(2+)-bound closed conformation and in two open Mg(2+)-free states at resolutions of 3.8, 7.1, and 7.1 Å, respectively. In the absence of bound Mg(2+), four of the five subunits are displaced to variable extents (∼ 10-25 Å) by hinge-like motions as large as ∼ 35° at the stalk helix. The transition between a single 5-fold symmetric closed state and an ensemble of low Mg(2+), open, asymmetric conformational states is, thus, the key structural signature of CorA gating. This mechanism is likely to apply to other structurally similar divalent ion channels.

Cryo-EM Analysis of the Conformational Landscape of Human P-glycoprotein (ABCB1) During its Catalytic Cycle

The multidrug transporter P-glycoprotein (P-gp, ABCB1) is an ATP-dependent pump that mediates the efflux of structurally diverse drugs and xenobiotics across cell membranes, affecting drug pharmacokinetics and contributing to the development of multidrug resistance. Structural information about the conformational changes in human P-gp during the ATP hydrolysis cycle has not been directly demonstrated, although mechanistic information has been inferred from biochemical and biophysical studies conducted with P-gp and its orthologs, or from structures of other ATP-binding cassette transporters. Using single-particle cryo-electron microscopy, we report the surprising discovery that, in the absence of the transport substrate and nucleotides, human P-gp can exist in both open [nucleotide binding domains (NBDs) apart; inward-facing] and closed (NBDs close; outward-facing) conformations. We also probe conformational states of human P-gp during the catalytic cycle, and demonstrate that, following ATP hydrolysis, P-gp transitions through a complete closed conformation to a complete open conformation in the presence of ADP.

Structural basis of kainate subtype glutamate receptor desensitization

Glutamate receptors are ligand-gated tetrameric ion channels that mediate synaptic transmission in the central nervous system. They are instrumental in vertebrate cognition and their dysfunction underlies diverse diseases. In both the resting and desensitized states of AMPA and kainate receptor subtypes, the ion channels are closed, whereas the ligand-binding domains, which are physically coupled to the channels, adopt markedly different conformations. Without an atomic model for the desensitized state, it is not possible to address a central problem in receptor gating: how the resting and desensitized receptor states both display closed ion channels, although they have major differences in the quaternary structure of the ligand-binding domain. Here, by determining the structure of the kainate receptor GluK2 subtype in its desensitized state by cryo-electron microscopy (cryo-EM) at 3.8 Å resolution, we show that desensitization is characterized by the establishment of a ring-like structure in the ligand-binding domain layer of the receptor. Formation of this ‘desensitization ring’ is mediated by staggered helix contacts between adjacent subunits, which leads to a pseudo-four-fold symmetric arrangement of the ligand-binding domains, illustrating subtle changes in symmetry that are important for the gating mechanism. Disruption of the desensitization ring is probably the key switch that enables restoration of the receptor to its resting state, thereby completing the gating cycle.

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.

Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase

Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an “open” or “closed” state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.

Using cryo-EM to Untangle the Conformational Landscape of a Small Allosterically-Regulated Complex

Use of direct-electron-detectors is driving an unprecedented increase in the resolution of protein complex structures solved by cryo-EM. Computer driven image acquisition combined with streamlined image processing pipelines offer the opportunity to determine structures relatively automated way. Here, we combine these developments to characterize the binding of small ligands on glutamate dehydrogenase (GDH), a clinically significant 336 kDa homohexameric enzyme that is a relevant pharmaceutical target for cancer, Parkinsons, and diabetes. Images from single specimens collected in a single session provided enough information to localize nucleotides in a complex at ∼3.2 A resolution. We first confirmed the validity of the approach by determining the structure of GDH complexes for which comparable crystallographic coordinates are available. We then found that binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture that can be resolved into two ∼3.3 A structures for which the enzyme is in either an “open” or “closed” state. The work-flow used in this work provides a streamlined path to rapidly solve the structure of macromolecular complexes, to image the binding target of drug molecules and to resolve conformers at near atomic resolution.