Hermann SchindelinPh.D., Free University Berlin
Postdoctoral Research, California Institute of Technology
Assistant Professor, Department of Biochemistry and Cell Biology
My lab uses crystallographic and biochemical techniques to study structural and functional aspects of biologically important proteins, mostly enzymes. Three different areas are currently investigated as outlined below. As part of the Center for Structural Biology we are located in the Centers for Molecular Medicine, a new research facility at Stony Brook. For more information and research opportunities visit the home page of the Schindelin lab.
1. Biosynthesis and function of the molybdenum cofactor
The molybdenum cofactor (Moco) is an essential component of a diverse group of enzymes catalyzing important redox transformations in the global carbon, nitrogen and sulfur cycles. In humans, the catalytic activities of three enzymes, sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase depend on this cofactor. The cofactor consists of a mononuclear molybdenum coordinated by the dithiolene moiety of a family of tricyclic pyrano-pterins containing a cis-dithiolene group in their pyran ring. This tricyclic pyranopterin is commonly referred to as molybdopterin (MPT) and the Mo-MPT complex as the molybdenum cofactor (Moco). Enzymes containing a pyranopterin cofactor and either Mo or W are found in all phyla and some of these catalyze important transformations in the global carbon, nitrogen and sulfur cycle. These reactions generally involve redox chemistry at the active site with the metal cycling between the +IV and +VI oxidation states.
Fig. 1: Schematic overview of the Moco biosynthetic pathway in E. coli.
Biosynthesis of the Mo/W-cofactor is an evolutionarily conserved pathway and genes involved in Moco biosynthesis have been identified in eubacteria, archaea and eukaryotes. Although some details of Moco biosynthesis are still unclear at present, the pathway can be divided into three phases as outlined in Fig. 1. (i) Early steps in which a guanosine derivative, most likely GTP, is converted into precursor Z. This aspect is different from other pterin biosynthetic pathways, since C8 of the purine is inserted between the 2' and 3' ribose carbon atoms during formation of precursor Z, rather than being eliminated. (ii) Transformation of precursor Z into molybdopterin. This process generates the dithiolene group responsible for coordination of the molybdenum atom in the cofactor, and the reaction is catalyzed by MPT synthase. MPT synthase consists of two subunits (MoaD and MoaE in E. coli) and in its active state the C-terminus of the small subunit is present as a thiocarboxylate. The activation of the small subunit is catalyzed by a sulfurtransferase (MoeB) in an ATP-dependent reaction reminiscent of the activation of ubiquitin by the ubiquitin-activating enzyme. (iii) Metal incorporation into the apo-cofactor. Based on the observation that high concentrations of molybdate in the growth medium can partially rescue a mogA mutant, MogA has been proposed to act as a molybdochelatase incorporating molybdenum into molybdopterin. MoeA is probably also involved in metal incorporation, possibly by converting molybdate into a thio-molybdenum containing compound. In addition to these conserved steps in the biosynthesis of the cofactor additional activities required for generating active cofactor exist in some organisms. For example, most enzymes from eubacteria contain a dinucleotide form of the cofactor in which a second nucleotide such as GMP or CMP is linked to the organic component of the cofactor via a pyrophosphate linkage.
We have determined or are in the process of determining the crystal structure of every protein shown in Fig. 1. The resulting models have provided initial insights into the function of these proteins. Of particular importance are the studies on the second and third step in this pathway, which transcend the field of Moco biosynthesis and have led us to the study of seemingly unrelated areas described in sections 2 and 3. To understand the mechanism of sulfur incorporation during the second step of Moco biosynthesis, we have determined the cocrystal structure of the MoeB-MoaD complex in its apo-form, and in complex with ATP. Due to the relationship of these proteins to ubiquitin-activation and the related processes of sumoylation and rubylation, as well as the sulfur incorporation during thiamine biosynthesis, these studies have provided not only insights into the mechanism of Moco biosynthesis, but also a structural basis for understanding these related processes. The structural studies of the MogA and MoeA proteins, which are involved in metal incorporation into the organic moiety of the cofactor, have provided us with structural models of the two building blocks of the multifunctional mammalian gephyrin protein. Gephyrin was initially isolated as being responsible for anchoring glycine receptors to the cytoskeleton at synaptic sites, but is also responsible for catalyzing molybdenum incorporation in mammals. For a more detailed description of the multiple roles of gephyrin see section 3. Our future aims in the field of Moco biosynthesis will focus on additional structural and biochemical studies, which aim to describe the step-by-step assembly of the Moco at atomic detail. In addition, we will try to structurally characterize the human orthologs of the bacterial proteins, since these provide better models for the analysis of mutations, which lead to Moco deficiency in humans.
2. Mechanisms of ubiquitin-dependent protein degradation
Proteolysis of cellular proteins is a highly complex, temporally controlled and tightly regulated pathway, which plays important roles in a broad array of basic cellular reactions. Defects in ubiquitin-mediated protein degradation have been shown to cause pathological conditions, including malignant transformations, abnormal immune and inflammatory responses as well as genetic and neurodegenerative diseases. A cascade of enzymes displaying a high degree of substrate specificity is involved in ubiquitin-dependent protein degradation. Among the cellular targets of this machinery are cell cycle and growth regulators, components of signal transduction pathways, enzymes of house-keeping and cell specific pathways as well as mutated or post-translationally damaged proteins. Degradation of the target proteins follows a two-step mechanism: (i) Covalent attachment of multiple ubiquitin molecules to the protein and (ii) its subsequent degradation by the 26S proteasome. Three enzymes are responsible for the covalent attachment of ubiquitin to a target molecule. In an ATP-dependent reaction the E1 component (ubiquitin activating enzyme) forms a high-energy thioester bond between a conserved cysteine in the E1 protein and the C-terminal glycine of ubiquitin. In the second reaction, the activated ubiquitin molecule is moved in a transesterification reaction to a cysteine of an E2 component (ubiquitin carrier protein or ubiquitin conjugating enzyme). Finally, ubiquitin is transferred to a lysine residue of the substrate molecule by the E3 component (ubiquitin ligase). This last step can be achieved either by direct binding of the substrate to E3 or binding via an adaptor molecule. Furthermore, some E3's first form a ubiquitin E3 complex, again via a cysteine residue, whereas other E3's transfer ubiquitin directly from the E2 component to the target protein. At present, four different families of E3 components have been characterized: (1) The main N-end rule E3's, (2) the HECT domain family, (3) the cyclosome or anaphase promoting complex (APC) and (4) the phosphoprotein-ubiquitin ligase complexes.
Our efforts in this area are currently focused on the crystal structure analyses of an E1 enzyme, the yeast UBA1 protein, and an E3-substrate complex, between the ubiquitin ligase Smurf1 (for Smad ubiquitination regulatory factor-1) and its cellular target Smad1. The work on UBA1 is an extension of our studies on the activation of MoaD by MoeB. We are trying to understand the molecular basis for the mechanistic differences between ubiquitin-activation and sulfur incorporation during Moco biosynthesis. Although the initial steps up to the formation of the acyladenylate are conserved between these two and other related systems, there are subsequent mechanistic differences, which lead to the formation of either a thioester during ubiquitin-activation or a thiocarboxylate during Moco biosynthesis. The studies on the Smurf1-Smad 1 complex are done in collaboration with my colleague Dr. Jerry Thompson. Using the yeast two hybrid system with Smad1 as bait, the Thompson lab has demonstrated a specific interaction between Smad1 and Smurf1. The Smad molecules are involved in signaling pathways triggered by the TGF-beta/Bone morphogenetic protein (BMP)/activin superfamily of cytokines. Downstream of the corresponding cytokine receptor, the Smad molecules mediate the signals from the plasma membrane to the nucleus and participate in a variety of cellular and developmental processes. Importantly, Smad2 and Smad4 have been identified as tumor suppressors in humans. The 80 kDa Smurf1 protein is an E3 enzyme and, based on sequence similarities in the C-terminal portion of the molecule, belongs to the HECT family of ubiquitin ligases. Members of this family bind directly to their target substrates and transfer the covalently attached ubiquitin from a conserved cysteine to the protein being targeted for degradation. Smurf1 specifically interacts with Smad1 and Smad5, two Smad proteins involved in BMP signaling, but appears to have little affinity towards Smad2 and 3, which are targeted by the receptors for activin and TGF-beta.
3. Understanding the anchoring of inhibitory neuronal receptors
One of the proteins involved in Moco biosynthesis in humans is gephyrin, which has been first identified as being responsible for the anchoring of glycinergic receptors to the cytoskeleton at inhibitory synapses. Gephyrin is able to simultaneously bind to the beta-subunit of the receptor and tubulin, thus linking the receptor to the cytoskeleton. In a related context, gephyrin has also been postulated to participate in the postsynaptic localization of major GABAA receptor subtypes. Gephyrin also interacts with the actin-binding protein profilin, and has been implicated in a signaling cascade induced by the immuno-suppressant rapamycin through its binding to RAFT1, a DNA-activating protein kinase. Finally, gephyrin was shown to interact with two alternative splice variants of collybistin, a newly identified brain-specific guanine nucleotide exchange factor. Several splice variants of gephyrin have been characterized, which could potentially contribute to the functional diversity of gephyrin.
As outlined above, gephyrin is a fusion protein of the E. coli MogA and MoeA proteins, which are both involved in the third step of Moco biosynthesis. The corresponding regions in gephyrin, here referred to as the G- and E-domains, respectively, are connected by a linker containing at least ~140 amino acids. In the most common splice variant, the human and rat gephyrin proteins contain 736 amino acids. The sequence similarities to the bacterial MogA and MoeA proteins suggested that gephyrin is also involved in Moco biosynthesis and, recently, a direct participation of gephyrin in Moco biosynthesis has been demonstrated. We have succeeded in the determination of the crystal structure of the E. coli MogA and MoeA proteins and are currently trying to crystallize holo-gephyrin as well as the individual E- and G-domains. The goal of this aspect of my research is to describe how the various functions of gephyrin are accomplished in the context of its three-dimensional structure. Specifically, we want to answer the following questions:
1. How are the active sites of the G- and E-domains arranged in the context of the three-dimensional structure of gephyrin, and is there any cooperativity between these active sites?
2. How is gephyrin able to act as an anchor for glycinergic receptors, and where are the binding sites for the beta-subunit of the glycinergic receptor and tubulin? What is the structural basis for gephyrin's involvment in rapamycin sensitive signaling? How does gephyrin interact with the two alternative splice variants of collybistin? How does the protein interact with the actin-binding protein profilin?
3. How are the different activities of gephyrin regulated by alternative splicing?
H. Schindelin, C. Kisker, J. Hilton, K.V. Rajagopalan and D.C. Rees "Crystal structure of DMSO reductase - Redox-linked changes in molybdopterin coordination", Science 272, 1615-1621 (1996). Abstract.
H. Schindelin, C. Kisker, J.L. Schlessman, J.B. Howard and D.C. Rees "Structure of ADP-AlF4--stabilized nitrogenase complex and its implications for signal transduction", Nature 387, 370-376 (1997). Abstract.
H. Schindelin, C. Kisker and D.C. Rees "The molybdenum cofactor: a crystallographic perspective", JBIC 2, 773-781 (1997). Abstract.
M.T.W. Liu, M.M. Wuebbens, K.V. Rajagopalan and H. Schindelin "Crystal structure of the gephyrin-related molybdenum cofactor biosynthesis protein MogA from Escherichia coli", J. Biol. Chem.275, 1814-1822 (2000). Abstract.
M.M. Wuebbens, M.T.W. Liu, K.V. Rajagopalan and H. Schindelin "Crystal structure of the molybdenum cofactor biosynthesis protein MoaC provides insights into molybdenum cofactor deficiency, Structure 8, 709-718 (2000). Abstract.
H.-K. Li, C. Temple, K.V. Rajagopalan and H. Schindelin "The 1.3 Å crystal structure of Rhodobacter sphaeroides dimethylsulfoxide reductase reveals two distinct molybdenum coordination environments", J. Am. Chem. Soc.122, 7673-7680 (2000). Abstract.
M.W. Lake, C.A. Temple, K.V. Rajagopalan and H. Schindelin "The Crystal Structure of the Escherichia coli MobA Protein Provides Insight into Molybdopterin Guanine Dinucleotide Biosynthesis", J. Biol. Chem. 275, 40211-40217 (2000). Abstract.
M.J. Rudolph, M.M. Wuebbens, K.V. Rajagopalan and H. Schindelin "Crystal structure of molybdopterin synthase and its evolutionary relationship to ubiquitin activation", Nature Struct. Biol, 8, 42-46 (2001). Abstract.
S.Xiang, J. Nichols, K.V. Rajagopalan and H. Schindelin. "The Crystal Structure of Escherichia coli MoeA and Its Relationship to the Multifunctional Protein Gephyrin", Structure 9, 299-310 (2001).