Post-translational and Post-transcriptional Ribosome Modification

Our structure determination of the PrmA methyltransferase in various protein-protein complexes with the substrate, ribosomal protein L11 provided the first exciting results for our exploration of ribosome modifications and the corresponding enzymes. To date, we have determined more than 14 different structures for PrmA and PrmA-L11 complexes (take a look at the crystals in our gallery).
In bacteria, the ribosomal protein L11 is post-translationally methylated at multiple sites by PrmA. PrmA is a highly unusual enzyme, because is modifies several side chains on the same substrate protein. Our studies show that the enzymes consists of a catalytic class I methyltransferase domain and an L11-specific substrate recognition domain. A highly flexible linker region connects both domains enabling the enzyme to place different substrate side chains into the active site cavity.

Hasan Demirci, Steven Gregory, Albert E. Dahlberg and Gerwald Jogl, Recognition of ribosomal protein L11 by the protein trimethyltransferase PrmA, EMBO Journal (2007) 26, 567-577. Pubmed

The PrmA – L11 complex structure with the substrate residue Lys39 in the active site.

In this study, we report further results for the enzymatic mechanism of the modification of protein L11 by PrmA. Here, we present complex structures with the N-terminal amino group of L11 placed in the PrmA active site. Most excitingly, we observed clear electron density for the N-terminal amino group in a fully trimethylated post-catalytic state. Together, our two PrmA publications provide important insights into the structural flexibility required for multiple methylation of the same substrate protein.

Demirci H, Gregory S, Dahlberg AE, Jogl G; Multiple-site trimethylation of ribosomal protein L11 by the PrmA methyltransferase.
Structure, 16, 1059-1066 (2008). Pubmed

The transition of protein L11 from its orientation with the N-terminal amino group placed in the active site to the orientation with K39 placed in the active site.

In this publication, we studied the RsmF methyltransferase from T. thermophilus. This enzyme modifies three bases in the 16S ribosomal RNA, which are located in distinct structural contexts in the ribosomal subunit. With biochemical studies, we showed that the enzyme prefers the 30S subunit to naked 16S rRNA as a substrate. Similar to our observations with PrmA and KsgA, we found that several key segments in the RsmF active site can assume different conformations in different crystal forms suggesting that these regions are dynamic in solution.

Demirci H, Larsen HGL, Hansen T, Rasmussen A, Cadambi A, Gregory S, Kirpekar F, Jogl G; Multi-site specific 16S rRNA methyltransferase RsmF from Thermus thermophilus.
RNA, 16, 1584-1596(2010). Pubmed

The cofactor-binding site in the RsmF crystal structure.

In this publication, we reported biochemical and structural studies of the KsgA methyltransferase. KsgA introduces the most highly conserved post-transcriptional modification in 16S ribosomal RNA. Analyzing the KsgA structure in different crystal forms, we identified several dynamic regions in the cofactor-binding site and in the likely substrate-recognition region. This supports our previous observations that multi-specific methyltransferases may require structural dynamics to modify several substrate sites.

Demirci H, Belardinelli R, Seri E, Gregory ST, Gualerzi C, Dahlberg AE, Jogl G; Structural rearrangements in the active site of the Thermus thermophilus 16S rRNA methyltransferase KsgA in a binary complex with 5'-methylthioadenosine.
J. Mol. Biol. 388, 271-282 (2009). Pubmed

Changes in the KsgA active site observed in two crystal forms.

In this publication, we report the structure of a mono-specific ribosomal RNA methyltransferase and biochemical studies describing the substrate specificity for this enzyme. This study represents the first step in a transition of our research focus from the ribosome-modifying enzymes to structural studies of ribosomal subunits. Loss of the modification introduced by RsmG results in resistance to the antibiotic streptomycin. The study of the molecular basis of this antibiotic resistance is a major current project in the laboratory.

Gregory ST, Demirci H, Belardinelli R, Monshupanee T, Gualerzi C, Dahlberg AE, Jogl G; Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG.
RNA 15, 1693-1704 (2009). Pubmed

Surface of RsmG colored by electrostatic potential.

The RsmC methyltransferase represents an example of a single substrate monomethyltransferases - the extreme opposite of the PrmA methyltransferase. In this study, we were able to determine a complex structure with a guanosine molecule bound in the active site, which mimics the placement of the substrate G1207 in 16S rRNA. This data enabled us to characterize the details of substrate base orientation and to better understand the coordination of the natural RNA substrate.

Demirci H, Gregory S, Dahlberg AE, Jogl G; Crystal structure of the Thermus thermophilus 16S rRNA methyltransferase RsmC in complex with cofactore and substrate guanosine.
J. Biol. Chem., 283, 26548-26556 (2008). Pubmed

Overall structure of RsmC illustrating the enzyme's two domain organization.

Structural Studies of the TIGAR phosphatase

The TIGAR enzyme is a newly identified fructose-2,6-bisphosphatase, which is activated by the p53 tumor suppressor protein and serves to downregulate cellular energy production in the glycolysis pathway. In this publication, we describe the biochemical and structural characterization of this enzyme. Two phosphate ions bound in the enzyme’s active site can be used as guidance to model the position of the substrate, fructose-2,6-bisphosphate.

See the Brown University Press Release

Li H & Jogl G; Structural and Biochemical Studies of TIGAR (TP53-Induced Glycolysis and Apoptosis Regulator).
J. Biol. Chem., 284, 1748-1454 (2009). Pubmed

Two phosphate ions bound in the active site of the TIGAR phosphatase.

Structural Basis of Inositol Signaling

Soluble inositol phosphates have been implicated to function as second messengers in essential cellular processes, and their levels are regulated by inositol phosphate kinases. In the first study of this project, we have determined the crystal structure of the yeast inositol phosphate multikinase Ipk2. The Ipk2 multikinase catalyzes two consecutive phosphorylations from inositol trisphosphate (IP3) to IP4 and from two different IP4 isomers to IP5 requiring an unusually flexible substrate recognition mechanism to accommodate these different substrates.
Our Ipk2 structure provided the first information for the inositol multikinase protein family, a subgroup of the inositol phosphate kinase superfamily. The overall structure of this enzyme is related to the more specific inositol phosphate 3-kinase. Especially the ATP-binding pocket is closely related and also homologous to other ATP-grasp protein kinases. The Ipk2 inositol-binding domain is unique and provides critical side chains in the inositol-binding site, which enables us to propose a model to rationalize the promiscuous substrate binding ability of this enzyme.

William Holmes and Gerwald Jogl, Crystal Structure of Inositol Phosphate Multikinase 2 and implications for substrate specificity, J. Biol. Chem. (2006) 281, 38109 -38116. Pubmed

Crystal structure of yeast inositol polyphosphate kinase Ipk2.

Crystal structure of the bacterial periplasmatic zinc transporter ZnuA

The zinc-specific transport protein ZnuA is the soluble, periplasmatic component of a bacterial metal uptake system and delivers zinc ions to an ATP-binding cassette transport system in the inner bacterial membrane. Several structures of zinc- and manganese-specific binding proteins revealed a conserved protein fold for metal binding. We have determined the crystal structure of ZnuA from E. coli to 1.75 A resolution. Similar to the other transport proteins, the zinc ion is bound in the interface between two structurally related globular domains. The metal ion is coordinated by three histidine residues and one glutamate residue representing a novel variation of metal coordination compared with the other available transporters. To further investigate the basis for discrimination between manganese and zinc in a structurally similar framework, we are currently performing biocalorimetry studies with site-directed ZnuA mutants.

Hua Li and Gerwald Jogl, Crystal structure of the zinc-binding transport protein ZnuA from Escherichia coli reveals an unexpected variation in metal coordination. J. Mol. Biol. (2007) 368, 1358-1366. Pubmed

2Fo-Fc electron density map for the active site of ZnuA

Biosynthesis of the pentalenolactone antibiotic: the crystal structure of the PtlH dioxygenase form Streptomyces avermitilis.
(in collaboration with the Cane Lab at Brown University)

The PtlH dioxygenase catalyzes an essential step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. PtlH is a member of the iron(II)/alpha-ketoglutarate dependent dioxygenase superfamily. We have determined the PtlH crystal structure to 1.37 A resolution in complex with the cofactors iron and alpha-ketoglutarate and the non-reactive substrate enantiomer ent-1-deoxypentalenic acid. The overall structure is a canonical double-stranded barrel helix fold similar to other dioxygenases. Additional secondary structure elements unique to PtlH provide a substrate binding site specific for the unusual tricyclic substrate. Using site-directed mutagenesis combined with enzyme kinetical studies, we have shown that two arginine residues in the active site are crucial for substrate binding and orientation. Computational docking studies based on our structures provided a structural model for the binding mode of the PtlH substrate.

Zheng You, Satoshi Omura, Harua Ikeda, David E. Cane, and Gerwald Jogl, Crystal Structure of the non-heme iron dioxygenase PtlH in pentalenolactone biosynthesis. J. Biol. Chem. Published online Nov. 2007. Pubmed

The non-reactive substrate enantiomer ent-1-deoxypentalenic acid bound in the active site of PtlH