12th Annual Fall Symposium

Advances in Structural Biology

Christopher W. Akey, Ph.D., Professor of Physiology and Biophysics, Boston University School of Medicine

The apoptosome and other signaling platforms. In the intrinsic death pathway, cytochrome c binds to Apaf-1 and triggers apoptosome assembly in the presence of dATP. This platform binds procaspase-9, which activates procaspase-3 and other downstream procaspases. To understand this process, we determined a structure of the human apoptosome at 12.8Å resolution and created a model of this heptameric platform, which contains 49 domains and 7 cytochrome c molecules. Higher resolution studies of the active apoptosome should provide additional insights into the assembly and function of this cell killer. In parallel studies, we are determining structures of the Drosophila Apaf-1 related killer (Dark) and of other signaling platforms that mediate inflammatory responses to bacteria.

Tim Baker, Professor, Department of Chemistry and Biochemistry, UCSD

Cryo-electron microscopy and 3D image reconstruction of viruses.The primary thrust of our research is to image viruses and gain insights about how they interact with their hosts, replicate, and mature. We use the tools of cryo-electron microscopy (CryoEM) and three-dimensional (3D), image-reconstruction to observe viruses or virus-like particles and examine intermolecular interactions at sub-nanometer resolutions. At such resolution, it is possible to discern and distinguish viral components (protein, nucleic acid, lipid, carbohydrate) and even visualize secondary structural details such as alpha-helices and beta-sheets in proteins. Viruses currently under investigation include bacteriophages phi-29 and P22; gemini viruses, several partitiviruses, parvoviruses (human bocavirus and several serotypes of adeno-associated viruses both as native virions and complexed with neutralizing antibodies and receptor molecules), reoviruses, a tetravirus, two distinct totiviruses, and several, large dsDNA viruses that infect algae and insects; and a family of small, enveloped, ssRNA viruses that include members that are serious human pathogens and have been listed as possible agents of bio-terrorism.

William Cramer, Henry Koffler Distinguished Professor of Biological Sciences, Purdue University

The Labyrinthine Internal Structure of the Photosynthetic Cytochrome b6f Complex. The hetero-oligomeric cytochrome bc complexes in photosynthetic and respiratory membranes catalyze transmembrane electron transfer coupled to proton translocation, which generates the trans-membrane proton electrochemical gradient utilized for synthesis of ATP. The dimeric b6f complex of oxygenic photosynthesis provides the electronic connection between the two photosynthetic reaction centers, the oxygen-evolving PSII and PSI linked to reduction of NADP+. The mechanism of proton translocation in this complex involves oxidation-reduction of the large lipophilic plastoquinol/quinone (PQH2/PQ) that contains a quinone ring and a tail containing 9 isoprenoid groups (45 carbons). The pathway within the protein complex through which the large quinone molecule travels and mediates oxidation-reduction and coupled proton transfer illustrates the labyrinthine nature of the internal structure of multi-subunit membrane proteins.

Martin Egli, Professor, Department of Biochemistry, Vanderbilt University School of Medicine.

Structure and Mechanism of the KaiABC Circadian Clock: Circadian clocks are self-sustained biochemical oscillators. Their properties include temperature compensation, a time constant of approximately 24 h, and high precision. Recent research has shown that the KaiABC circadian clock from the cyanobacterium S. elongatus can be reconstituted in vitro from the three proteins KaiC, KaiA and KaiB in the presence of ATP. This renders the KaiABC molecular timer a unique target of biochemical and biophysical studies. We are characterizing this clock system using X-ray crystallography in combination with electron microscopy and mutagenetic studies. We have recently determined the crystal structure of the KaiC auto-kinase and auto-phosphatase and have presented three-dimensional models of binary KaiA-KaiC and KaiB-KaiC complexes that shed light on the roles of the KaiA and KaiB proteins in controlling the KaiC phosphorylation status.

Brandt Eichman, Associate Professor, Departments of Biological Sciences and Biochemistry, Vanderbilt University

Molecular mechanism of eukaryotic DNA replication initiation. The initiation of DNA replication in eukaryotic cells is a highly regulated process that is essential for maintenance of genome integrity. Failure to copy the genome only once and at the proper time during the cell cycle can lead to elevated mutation rates, chromosome instability, and the development of cancer. This process involves a choreographed assembly of several dynamic protein complexes which must recognize and unwind DNA at origins of replication, interpret cell cycle signals, and ultimately result in the formation of active replication forks. We are currently working to determine the crystal structures of several initiation proteins which are required for DNA unwinding and loading of DNA polymerases. Using a combination of structural, biophysical, biochemical, and biological approaches, we aim to generate a comprehensive model for the spatial arrangement of these proteins during DNA unwinding in an effort to understand the mechanism of replication initiation.

Tom Ellenberger, Wittcoff Professor and Head, Department of Biochemistry and
Molecular Biophysics, Washington University in St. Louis

The Structural Cell Biology of DNA Repair. DNA excision repair proteins recognize chemically modified nucleotides and catalyze their removal from DNA. We are investigating the mechanisms of base-flipping and catalysis by DNA glycosylases through biochemical assays and crystallographic structure determinations. Our goal is to understand how these enzymes select damaged nucleotides in a vast excess of normal DNA. Bulky chemical adducts are excised from DNA by a large complex of nucleases and DNA remodeling enzymes formed during nucleotide excision repair (NER). We are investigating how the physical interactions of NER proteins specify the incision of the DNA backbone on either side of the damage.

Elizabeth Getzoff, Professor, Department of Molecular Biology
and The Skaggs Institute for Chemical Biology, Scripps Research Institute

Structural Mechanism of Abscisic Acid Binding and Signaling by Dimeric PYR1. The phytohormone abscisic acid (ABA) acts in seed dormancy, plant development, drought tolerance, and adaptive responses to environmental stresses. Structural mechanisms mediating ABA receptor recognition and signaling remain unknown, but are essential for understanding and manipulating abiotic stress resistance. Here, we report structures of PYR1, a prototypical PYR/PYL/RCAR protein that functions in early ABA signaling. The crystallographic structure reveals an / helix-grip fold and homodimeric assembly, verified in vivo by co-immunoprecipitation. ABA binding within a large internal cavity switches structural motifs distinguishing ABA-free "open-lid" from ABA-bound "closed-lid" conformations. Small angle x-ray scattering suggests that ABA signals by converting PYR1 to a more compact, symmetric closed-lid dimer. Site-directed PYR1 mutants designed to disrupt hormone binding lose ABA-triggered interactions with type 2C protein phosphatase partners in planta.

Joe Jez, Assistant Professor, Biology Department, Washington University in Saint Louis.

Sensing sulfur in plants: biochemical integration of multiple inputs. Sulfur is essential for plant growth and development, and the molecular systems for maintaining sulfur and thiol metabolism are tightly controlled. From a biochemical perspective, the regulation of plant thiol metabolism highlights nature’s ability to engineer pathways that respond to multiple inputs and cellular demands under a range of conditions. The mechanisms forming the molecular basis of biochemical sulfur sensing in plants range from the simple (substrate availability, thermodynamic properties of reactions, feedback inhibition, and organelle localization) to the elaborate (formation of multienzyme complexes and thiol-based redox switches). Ultimately, the dynamic interplay of these regulatory systems is critical for sensing and maintaining sulfur assimilation and thiol metabolism in plants.

Jan Kern, Lawrence Berkeley National Laboratory, University of California

The Structure of photosystem II. Photosystem II (PSII) is a large homodimeric protein–cofactor complex located in the photosynthetic thylakoid membrane that acts as light-driven water:plastoquinone oxidoreductase. The crystal structure of PSII from Thermosynechococcus elongatus at 2.9-Å resolution allowed the unambiguous assignment of all 20 protein subunits and complete modeling of all 35 chlorophyll a molecules and 12 carotenoid molecules, 25 integral lipids and 1 chloride ion per monomer. The presence of a third plastoquinone QC and a second plastoquinone-transfer channel, which were not observed before, suggests mechanisms for plastoquinol-plastoquinone exchange, and we calculated other possible water or dioxygen and proton channels. Putative oxygen positions obtained from a Xenon derivative indicate a role for lipids in oxygen diffusion to the cytoplasmic side of PSII. The chloride position suggests a role in proton-transfer reactions because it is bound through a putative water molecule to the Mn4Ca cluster at a distance of 6.5 Å and is close to two possible proton channels.

Joe Noel, Professor, Jack H. Skirball Center for Chemical Biology and Proteomics & Howard Hughes Medical Institute, Salk Institute for Biological Studies.

Mechanistic, Structural and Evolutionary Basis for Chemical Complexity in Nature. The focus of the research in our laboratory is to decipher the core principles influencing evolutionary change in proteins and protein networks particularly enzymes and metabolic pathways underlying the emergence and rapid expansion of chemical diversity in living systems. We ultimately hope to understand the chemical, structural and evolutionary tenets governing this extraordinary form of biodiversity and biocomplexity. In addition to probing the fundamental nature of molecular evolution, we also aim to exploit what we learn to direct our efforts at harnessing and altering these pathways to generate chemical "scaffolds" for the development of small molecule tools modulating proteins, cells and organisms.

John Peters, Professor, Montana State University.

Insights into the biosynthesis and evolution of complex iron-sulfur clusters containing hydrogenase and nitrogenase. Complex enzymes containing Fe–S clusters are ubiquitous in nature, where they are involved in a number of fundamental processes including carbon dioxide fixation, nitrogen fixation and hydrogen metabolism1,2. Hydrogen metabolism is facilitated by the activity of three evolutionarily and structurally unrelated enzymes: the [NiFe]- hydrogenases, [FeFe]-hydrogenases and [Fe]-hydrogenases3,4 (Hmd). The catalytic core of the [FeFe]-hydrogenase (HydA), termed the H-cluster, exists as a [4Fe–4S] subcluster linked by a cysteine thiolate to amodified 2Fe subcluster with unique non-protein ligands5,6. The 2Fe subcluster and non-protein ligands are synthesized by the hydrogenase maturation enzymes HydE, HydF and HydG; however, the mechanism, synthesis and means of insertion of H-cluster components remain unclear7–10. Here we show the structure of HydADEFG (HydA expressed in a genetic background devoid of the active site H-cluster biosynthetic genes hydE, hydF and hydG) revealing the presence of a [4Fe–4S] cluster and an open pocket for the 2Fe subcluster. The structure indicates that H-cluster synthesis occurs in a stepwise manner, first with synthesis and insertion of the [4Fe–4S] subcluster by generalized host-cell machinery11,12 and then with synthesis and insertion of the 2Fe subcluster by specialized hydE-, hydF- and hydG-encoded maturationmachinery7–10. Insertion of the 2Fe subcluster presumably occurs through a cationically charged channel that collapses following incorporation, as a result of conformational changes in two conserved loop regions. The structure, together with phylogenetic analysis, indicates that HydA emerged within bacteria most likely from a Nar1-like ancestor lacking the 2Fe subcluster, and that this was followed by acquisition in several unicellular eukaryotes.

Thomas Smith, Member and Principal Investigator, Donald Danforth Plant Science Center

Glutamate Dehydrogenase and Insulin Disorders. Mammalian glutamate dehydrogenase (GDH) is a mitochondrial enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate using NAD(P)+ as coenzyme. The enzyme is a homohexamer that is tightly regulated by a large number of positive and negative allosteric effectors as well as by cooperative interactions between subunits. While the enzyme is found in all organisms, this regulation is only found in the animal form. In collaboration with Dr. Charles Stanley's laboratory, we found that this regulation of GDH in mammals plays a crucial role in insulin homeostasis. This has been made evident by hyperinsulinism/hyperammonemia that is caused by a loss in the GTP inhibitory site of GDH. We have been using a variety of methods to identify the root cause of the disorder and to design potential therapeutics for this and other insulin disorders. Our current and most promising lead drugs are two compounds found in green tea, ECG and EGCG.

Richard Vierstra, Professor of Genetics, University of Wisconsin.

Structural basis for the photoconversion of a phytochrome to the activated Pfr form. Phytochromes are a collection of bilin-containing photoreceptors that regulate numerous photoresponses in plants and microorganisms through their ability to photointerconvert between a red-light-absorbing, ground state (Pr) and a far-red-light-absorbing, photoactivated state (Pfr). Although the structures of several phytochromes as Pr have been determined, little is known about the structure of Pfr and how it initiates signalling. We have determined the three-dimensional solution structure of the bilin-binding domain as Pfr, using the cyanobacterial phytochrome from Synechococcus OSB′. Contrary to predictions, light-induced rotation of the A pyrrole ring but not the D ring is the primary motion of the chromophore during photoconversion. Subsequent rearrangements within the protein then affect intradomain and interdomain contact sites within the phytochrome dimer. On the basis of our models, we propose that phytochromes act by propagating reversible light-driven conformational changes in the bilin to altered contacts between the adjacent output domains, which in most phytochromes direct differential phosphotransfer.

Thomas Walz, Professor, Harvard Medical School.

Membrane Pores and Channels. Biological membranes form a semi-permeable barrier surrounding all living cells. The types of molecules that can enter or exit the cell are defined by the integral proteins embedded in the membrane, which have very specific substrate specificities and are often regulated. Membrane proteins either form a pore or a channel, which allow the passive diffusion of a molecule along its concentration gradient, or they constitute energy-dependent pumps or transporters, which transport molecules against their concentration gradient. To fully understand the function of a membrane protein, knowledge of its structure is absolutely essential. Only then can we hope to resolve questions concerning substrate specificity, regulation and energy coupling. Although an estimated third of all proteins are membrane proteins, only a very limited number of membrane protein structures are available. This is due to the amphipathic nature of membrane proteins, which poses unique problems to determining their structure by X-ray crystallography and NMR spectroscopy. Electron microscopy is a viable alternative to these more established techniques to visualize the structure of membrane proteins. Electron crystallography of two-dimensional (2D) crystals can achieve near-atomic resolution and has already been used to determine the structure of a number of membrane proteins. We have substantial experience in growing 2D crystals and we attempt to use electron crystallography to determine the structure of a membrane protein whenever we have sufficient quantities to perform 2D crystallization trials. Recently we became interested in studying the interactions of membrane proteins with their surrounding lipids, for which electron crystallography is particularly well suited. We also use single particle electron microscopy to study gross conformational changes in membrane proteins related to their function and to visualize complexes formed by a membrane protein with other proteins.

Todd Yeates, Professor, UCLA

Structure of supramolecular assemblies. The Yeates laboratory is located at UCLA in the Department of Chemistry and Biochemistry. Our research covers the areas of molecular, structural and computational biology. In structural biology, our emphasis is on supramolecular protein assemblies, such as self-assembling protein filaments, layers, and cages. Supramolecular assemblies of interest include both natural and designed structures. Much of our current focus is on bacterial microcompartments, which are giant virus-like structures that serve as primitive organelles inside many bacterial cells. We have recently determined the three-dimensional atomic structures of several proteins that make up the shell of the carboxysome, a microcompartment that performs CO2 fixation, providing the first detailed clues about how it operates. Strategies are also being developed for designing novel proteins to assemble into ordered structures on the mid-nanometer scale. In the areas of computational biology and bioinformatics, our recent studies have led to the discovery of unusual microbes that are able to stabilize their proteins at extreme temperatures through mechanisms such as widespread disulfide bonding, and the use of topological complexity like linking and knotting. We also continue to work on developing new methods for deciphering protein function and protein interactions by applying ideas in symbolic logic to genomic data.

Ning Zheng, Professor, Howard Hughes Medical Institute /University of Washington

Protein Ubiquitination in Eukaryotic Biology. Ubiquitin is a small protein conserved in all eukaryotes. Eukaryotic cells use ubiquitin to modify many other proteins to modulate their functions. When a protein substrate is modified by a chain of multiple ubiquitin molecules, it will be targeted to the proteasome for degradation. Such a ubiquitin-dependent protein degradation mechanism plays a central role in regulating diverse cellular processes. The Zheng lab uses the structural biology approach to study multi-subunit enzyme machinery involved in protein ubiquitination. Our ongoing projects focus on ubiquitination events associated with human diseases such as cancer development and viral infection, as well as basic biology including hormone signaling and transcriptional control through the protein ubiquitination pathways in plants and yeast.