Research
The Spiegel Research Group is concerned with studying the structure, stability and function of various biochemical processes. In order to understand the complexities of each system, we employ techniques such as X-ray crystallography, small angle X-ray scattering (SAXS), fluorescence and other spectroscopic approaches, and traditional protein and nucleic acid biochemistry and biophysical approaches.
Structural and Functional Studies of Ribosomal GTPases
Ribosomal Translocation and Associated GTPases
During protein synthesis, or ‘translation’, messenger RNA (mRNA) and transfer RNA (tRNA) move in a coordinated manner through ribosomes to accurately synthesize defined polypeptides. This coordinated movement correlates with the ratchet-like rotation of the 30S subunit relative to the 50S subunit, indicating this movement is central to ribosomal function. Concomitant with intersubunit ratcheting is the iterative movements of tRNA through the ribosome via the “hybrid states” model of translocation. Complete, rapid, and accurate translocation is catalyzed at in vivo rates by the ribosomal GTPase, elongation factor G (EF-G). The view of ribosomal translocation has been complicated further by the newly described ribosomal back-translocase, elongation factor 4 (EF4, also called LepA), which can catalyze the reverse movement of ribosome-bound tRNA from the post- to pre-translocational states in a GTP-dependent manner. Structural comparison of EF-G and EF4 indicate that most of their structures are conserved with the exception of unique domains that protrude into the ribosomal A site (Figure 1). For the past six years my research program has been actively studying the ribosomal components required for GTPase activation, the role of thiopeptide antibiotics in GTPase inactivation, and the structural mechanism of EF4-catalyzed reverse translocation.
The Mechanism of GTPase Inactivation by Thiostrepton
Thiostrepton, a macrocyclic thiopeptide antibiotic, inhibits prokaryotic translation by interfering with the function of elongation factor G (EF-G). In this study, we have used 70S ribosome binding and GTP hydrolysis assays to study the effects of thiostrepton on EF-G and the newly described translation factor, elongation factor 4 (EF4). In the presence of thiostrepton, ribosome-dependent GTP hydrolysis is inhibited for both EF-G and EF4, with IC50 values equivalent to the 70S ribosome concentration (0.15 µM). Further studies indicate that the mode of thiostrepton inhibition is to abrogate the stable binding of EF-G and EF4 to the 70S ribosome (Figure 2). In support of this hypothesis, an EF-G truncation variant that does not possess domains IV and V was shown to possess ribosome-dependent GTP hydrolysis activity that was not affected by the presence of thiostrepton (>100 µM). The results of this study definitively indicate that the physiological mechanism of thiostrepton is to inhibit binding, and thus GTP hydrolysis and concurrent function, of GTPases that possess a domain V homologous to EF-G, which is in contrast to previous studies that suggest that thiostrepton does not inhibit GTP hydrolysis, but EF-G release from the ribosome.
Structural Mechanism of EF4-Catalyzed Reverse Translocation
Recently, it was shown that a conserved ribosomal GTPase, EF4 (also named LepA), is required for efficient and accurate translocation. It was further shown that EF4 possesses the unique function of reverse translocating the ribosome from the post- to the pre-translocation state. One crucial aspect of the process of reverse translocation that has not been described yet is how this occurs structurally. Recent studies in my research group have shown that EF4 stabilizes the ratcheted state of the 70S ribosome in the presence of non-hydrolyzable GTP (GDPNP). This complex is unique in that it is not coincident with the stabilization of the hybrid P/E state as was previously described for EF-G. Based on our structural data, along with the observation that E-site tRNA is required for reverse translocation, we propose a ‘retro-hybrid states’ model of reverse movement of tRNA (Figure 3) as described in our recent publication (Walter, et. al. (2012)).
Figure 1: Superposition of EF-G and EF4
Structural alignment reveals strong structural homology between domain V of EF-G (red) and the homologous domain in EF4 (green). The remainder of EF-G and EF4 is colored blue and grey, respectively.
Figure 2. Thiostrepton inhibition of ribosome-dependent multiple-turnover GTP hydrolysis.
(A) EF-G and (B) EF4. Samples consisted of GTPase + ribosomes (●), GTPase + ribosomes + thiostrepton (○), GTPase alone (n), or ribosomes alone (▲). Structure figure: Alignment of the 50S•thio and 70S•EF-G x-ray crystal structures.
Figure 3. ‘Hybrid States’ Model of Reverse Translocation. Upon proper forward translocation catalyzed by EF-G/GTP, E-site tRNA is released following GTP hydrolysis by EF-G. Improper translocation (which increases in frequence at low temperatures and high Mg2+ concentrations) results in E-site tRNA remaining bound to the 70S ribosome. Reverse translocation is then energetically favored, and may occur spontaneously or catalyzed by EF4 in a GTP-dependent manner.
Structural Studies of Factor VIII to Overcome the Immune Response
Factor VIII is an essential, non-enzymatic cofactor that performs a central role in the regulation of blood coagulation. Using a combination of biochemical, thermodynamic and crystallographic approaches, we are attempting to (A) generate novel biochemical reagents that improve the existing therapeutic molecules for use as hemophilia replacement therapy; and (B) study the immune response the is activated upon the introduction of factor VIII to a severe hemophiliac. The central component of this project is the factor VIII C2 domain, which is responsible for binding activated platelets and recruiting coagulation factors to the site(s) of vascular injury.
Structure and Stability Studies of the Factor VIII C2 Domain
A long known issue with factor VIII physiology is that the active protein is inherently labile and possesses a short half-life in circulation, making it difficult to produce and administer for therapeutic purposes to hemophilia patients. Previous studies showed that porcine factor VIII is stable in circulation in comparison to the human sequence and has been shown to be able to overcome the action of antibody inhibitors. In order to determine the role of folding stability in factor VIII activity, we have been working towards characterizing the stability of the porcine and human sequences of the C2 domain, which is a functionally essential region of factor VIII. Presently, we have made initial characterization of the thermal and chemical stabilities of both the human and porcine C2 sequences, and have found that the porcine sequence is modestly more stable. Furthermore, we are also attempting to determine the x-ray crystal structure of the porcine sequence to determine its x-ray crystal structure at atomic resolution. We have recently collected diffraction data to 1.7 Å resolution and are currently refining the structure.
Figure 4: Structural studies of the FVIII C2 domain in a ternary complex with the inhibitory antibodies, 3E6 and G99.
(A) Molecular surface envelope generated from small angle x-ray scattering data for the ternary complex, indicating an extended structure with epitopes on opposing sides of the FVIII C2 domain.
(B) Preliminary model of ternary complex from x-ray crystallographic data. This model is the molecular replacement solution of the ternary complex with the CDR loops removed. Upon completion of the structure this summer, well-defined “classical” and “non-classical” epitopes will be described.
Structural Characterization of Factor VIII C2 Domain/Antibody Complexes
Therapy for hemophilia A is most commonly infusion of either plasma-derived or recombinant factor VIII. Today, the most significant complication of factor VIII replacement therapy is the development of factor VIII inhibitor antibodies, appearing in approximately 30% of patients with hemophilia A. The majority of these antibody inhibitors targets either the C2 or A2 domains of factor VIII, and commonly disrupts the function of the cofactor in vitro. We are currently studying two antibodies that specifically interact with the human C2 domain. One of them, 3E6, is a classical antibody that blocks the ability of factor VIII to associate with negatively charged membrane surfaces. The second antibody, G99, is a ‘non-classical’ antibody that disrupts the proteolytic activation of factor VIII by thrombin. Together, these two antibodies recognize two distinct epitopes within the C2 domain and block to procoagulant function of factor VIII cooperatively. Currently, we have shown that Fab fragments from each antibody binds to the isolated C2 domain and we have successfully formed the ternary complex (C2/3E6/G99) as measured by size exclusion chromatography, affinity pull-down assays, and ELISA methods. Furthermore, we have studied each of these complexes by both small angle x-ray scattering (SAXS) and x-ray crystallographic methods. We have recently determined the crystal structure of the C2 domain in a ternary complex with both antibodies to 2.47 Å resolution, which allows for direct observation of each antibody epitope (Figure 4).
Structural Studies of Engineered Proteins and Protein Complexes
The next generation of therapeutic and biotechnological molecules will likely be developed from the world of computational protein design. While these methods are still in development, the limitations of such techniques are often within the experimental structural validation of such proteins and protein complexes. Our research group has been active in establishing collaborations with other investigators to validate a number of these computationally designed protein complexes.
Structure Determination of a Computationally Engineered Protein-Protein Heterodimer
We recently collaborated with David Baker’s lab at the University of Washington to determine the x-ray crystal structures of newly engineered protein-protein complexes. We successfully expressed, purified, and crystallized each of the proteins in the complex. This allowed us to solve the structure of one of the computationally designed proteins to 2.0 Å resolution (Figure 5), and the other to 3.8 Å resolution (not refined). Our data indicated that subsequent to altering much of the sequence of each protein, their overall tertiary structures remained intact.
I-MsoI Mutants with Non-specific Activities Bound to DNA Target Site Variants
Additionally, we have completed an additional project with David Baker’s group to solve homing endonuclease-DNA complexes that have been engineered to display altered specificity. We were able to successfully solve the x-ray crystal structures of six different protein/DNA complexes that possess broad or altered specificity in relation to the wild type protein and DNA sequences. Generally, we have found that loss of base-specific contacts within certain regions of the I-MsoI target site does not dramatically affect the cleavage activity, but can be tailored to changes, and in some cases decreases, in specificity (Figure 6). These new mutants of I-MsoI can potentially be used as alternative starting models for engineering more absolute changes in base specificity to tailor gene-specific reagents. We’re currently putting these structures in the context of their cleavage activities and preparing a manuscript for publication.
Figure 5. Structural Alignment of Designed Protein Scaffolds with Crystal Structures.
A structure of the Prb protein (magenta, solved in the Spiegel research group) agrees well with the design model (yellow, 0.54A ̊ all-atom rmsd) and structures of the PH1109 scaffold either apo (light green, 0.41A ̊ all-atom rmsd) or bound to CoA (dark green, 0.47A ̊ all-atom rmsd).
Figure 6. Structure and Activity Analysis of I-MsoI Variants.
Structure figure (left) indicates overlay between wild type and K28T variant shows change in H-bonding pattern at DNA position ±6. Activity figure (right) indicates that the K28T variant maintains activity but loses specificity.
