Marcus D. Hartmann

Structure and Activity of the N-Terminal Substrate Recognition Domains in Proteasomal ATPases

The proteasome forms the core of the protein quality control system in archaea and eukaryotes and also occurs in one bacterial lineage, the Actinobacteria. Access to its proteolytic compartment is controlled by AAA ATPases, whose N-terminal domains (N domains) are thought to mediate substrate recognition. The N domains of an archaeal proteasomal ATPase, Archaeoglobus fulgidus PAN, and of its actinobacterial homolog, Rhodococcus erythropolis ARC, form hexameric rings, whose subunits consist of an N-terminal coiled coil and a C-terminal OB domain. In ARC-N, the OB domains are duplicated and form separate rings. PAN-N and ARC-N can act as chaperones, preventing the aggregation of heterologous proteins in vitro, and this activity is preserved in various chimeras, even when these include coiled coils and OB domains from unrelated proteins. The structures suggest a molecular mechanism for substrate processing based on concerted radial motions of the coiled coils relative to the OB rings.

A Divergent Sm Fold in EDC3 Proteins Mediates DCP1 Binding and P-Body Targeting

ABSTRACT Members of the (L)Sm (Sm and Sm-like) protein family are found across all kingdoms of life and play crucial roles in RNA metabolism. The P-body component EDC3 (enhancer of decapping 3) is a divergent member of this family that functions in mRNA decapping. EDC3 is composed of a N-terminal LSm domain, a central FDF domain, and a C-terminal YjeF-N domain. We show that this modular architecture enables EDC3 to interact with multiple components of the decapping machinery, including DCP1, DCP2, and Me31B. The LSm domain mediates DCP1 binding and P-body localization. We determined the three-dimensional structures of the LSm domains of Drosophila melanogaster and human EDC3 and show that the domain adopts a divergent Sm fold that lacks the characteristic N-terminal α-helix and has a disrupted β4-strand. This domain remains monomeric in solution and lacks several features that canonical (L)Sm domains require for binding RNA. The structures also revealed a conserved patch of surface residues that are required for the interaction with DCP1 but not for P-body localization. The conservation of surface and of critical structural residues indicates that LSm domains in EDC3 proteins adopt a similar fold that has separable novel functions that are absent in canonical (L)Sm proteins.

A Coiled-Coil Motif that Sequesters Ions to the Hydrophobic Core.

Most core residues of coiled coils are hydrophobic. Occasional polar residues are thought to lower stability, but impart structural specificity. The coiled coils of trimeric autotransporter adhesins (TAAs) are conspicuous for their large number of polar residues in position d of the core, which often leads to their prediction as natively unstructured regions. The most frequent residue, asparagine (N@d), can occur in runs of up to 19 consecutive heptads, frequently in the motif [I/V]xxNTxx. In the Salmonella TAA, SadA, the core asparagines form rings of interacting residues with the following threonines, grouped around a central anion. This conformation is observed generally in N@d layers from trimeric coiled coils of known structure. Attempts to impose a different register on the motif show that the asparagines orient themselves specifically into the core, even against conflicting information from flanking domains. When engineered into the GCN4 leucine zipper, N@d layers progressively destabilized the structure, but zippers with 3 N@d layers still folded at high concentration. We propose that N@d layers maintain the coiled coils of TAAs in a soluble, export-competent state during autotransport through the outer membrane. More generally, we think that polar motifs that are both periodic and conserved may often reflect special folding requirements, rather than an unstructured state of the mature proteins.

A Widespread Glutamine-Sensing Mechanism in the Plant Kingdom

Glutamine is the primary metabolite of nitrogen assimilation from inorganic nitrogen sources in microorganisms and plants. The ability to monitor cellular nitrogen status is pivotal for maintaining metabolic homeostasis and sustaining growth. The present study identifies a glutamine-sensing mechanism common in the entire plant kingdom except Brassicaceae. The plastid-localized PII signaling protein controls, in a glutamine-dependent manner, the key enzyme of the ornithine synthesis pathway, N-acetyl-l-glutamate kinase (NAGK), that leads to arginine and polyamine formation. Crystal structures reveal that the plant-specific C-terminal extension of PII, which we term the Q loop, forms a low-affinity glutamine-binding site. Glutamine binding alters PII conformation, promoting interaction and activation of NAGK. The binding motif is highly conserved in plants except Brassicaceae. A functional Q loop restores glutamine sensing in a recombinant Arabidopsis thaliana PII protein, demonstrating the modular concept of the glutamine-sensing mechanism adopted by PII proteins during the evolution of plant chloroplasts.

The Structure of E. coli IgG-Binding Protein D Suggests a General Model for Bending and Binding in Trimeric Autotransporter Adhesins

The Escherichia coli Ig-binding (Eib) proteins are trimeric autotransporter adhesins (TAAs) and receptors for IgG Fc. We present the structure of a large fragment of the passenger domain of EibD, the first TAA structure to have both a YadA-like head domain and the entire coiled-coil stalk. The stalk begins as a right-handed superhelix, but switches handedness halfway down. An unexpected β-minidomain joins the two and inserts a ∼120° rotation such that there is no net twist between the beginning and end of the stalk. This may be important in folding and autotransport. The surprisingly large cavities we found in EibD and other TAAs may explain how TAAs bend to bind their ligands. We identified how IgA and IgG bind and modeled the EibD-IgG Fc complex. We further show that EibD promotes autoagglutination and biofilm formation and forms a fibrillar layer covering the cell surface making zipper-like contacts between cells.

Complete Fiber Structures of Complex Trimeric Autotransporter Adhesins Conserved in Enterobacteria.

Trimeric autotransporter adhesins (TAAs) are modular, highly repetitive surface proteins that mediate adhesion to host cells in a broad range of Gram-negative pathogens. Although their sizes may differ by more than one order of magnitude, they all follow the same basic head-stalk-anchor architecture, where the head mediates adhesion and autoagglutination, the stalk projects the head from the bacterial surface, and the anchor provides the export function and attaches the adhesin to the bacterial outer membrane after export is complete. In complex adhesins, head and stalk domains may alternate several times before the anchor is reached. Despite extensive sequence divergence, the structures of TAA domains are highly constrained, due to the tight interleaving of their constituent polypeptide chains. We have therefore taken a “domain dictionary” approach to characterize representatives for each domain type by X-ray crystallography and use these structures to reconstruct complete TAA fibers. With SadA from Salmonella enterica, EhaG from enteropathogenic Escherichia coli (EHEC), and UpaG from uropathogenic E. coli (UPEC), we present three representative structures of a complex adhesin that occur in a conserved genomic context in Enterobacteria and is essential in the infection process of uropathogenic E. coli. Our work proves the applicability of the dictionary approach to understanding the structure of a class of proteins that are otherwise poorly tractable by high-resolution methods and provides a basis for the rapid and detailed annotation of newly identified TAAs.

Crystallographic snapshot of the Escherichia coli EnvZ histidine kinase in an active conformation

Sensor histidine kinases are important sensors of the extracellular environment and relay signals via conformational changes that trigger autophosphorylation of the kinase and subsequent phosphorylation of a response regulator. The exact mechanism and the regulation of this protein family are a matter of ongoing investigation. Here we present a crystal structure of a functional chimeric protein encompassing the entire catalytic part of the Escherichia coli EnvZ histidine kinase, fused to the HAMP domain of the Archaeoglobus fulgidus Af1503 receptor. The construct is thus equivalent to the full cytosolic part of EnvZ. The structure shows a putatively active conformation of the catalytic domain and gives insight into how this conformation could be brought about in response to sensory input. Our analysis suggests a sequential flip-flop autokinase mechanism.

Crystal structure of a dimeric archaeal cleavage and polyadenylation specificity factor.

Proteins of the metallo-β-lactamase (MβL) fold form a large superfamily of metallo-hydrolase/oxidoreductases. Members of this family are found in all three domains of life and are involved in a variety of biological functions related to hydrolysis, redox processes, DNA repair and uptake, and RNA processing. We classified the archaeal homologs of this superfamily based on sequence similarity and characterized a subfamily of the Cleavage and Polyadenylation Specificity Factor (CPSF) with an uncommon domain composition: in addition to an extended MβL domain, which accommodates the active site for RNA cleavage, this group has two N-terminal KH domains. Here, we present the crystal structure of a member of this group from Methanosarcina mazei. It reveals a dimerization mode of the MβL domain that has not been observed before and suggests that RNA is bound across the dimer interface, recognized by the KH domains of one monomer, and cleaved at the active site of the other.

Thalidomide Mimics Uridine Binding to an Aromatic Cage in Cereblon.

Thalidomide and its derivatives lenalidomide and pomalidomide are important anticancer agents but can cause severe birth defects via an interaction with the protein cereblon. The ligand-binding domain of cereblon is found, with a high degree of conservation, in both bacteria and eukaryotes. Using a bacterial model system, we reveal the structural determinants of cereblon substrate recognition, based on a series of high-resolution crystal structures. For the first time, we identify a cellular ligand that is universally present: we show that thalidomide and its derivatives mimic and compete for the binding of uridine, and validate these findings in vivo. The nature of the binding pocket, an aromatic cage of three tryptophan residues, further suggests a role in the recognition of cationic ligands. Our results allow for general evaluation of pharmaceuticals for potential cereblon-dependent teratogenicity.

A domain dictionary of trimeric autotransporter adhesins.

Trimeric autotransporter adhesins (TAAs) are modular, highly repetitive outer membrane proteins that mediate adhesion to external surfaces in many Gram-negative bacteria. In recent years, several TAAs have been investigated in considerable detail, also at the structural level. However, in their vast majority, putative TAAs in prokaryotic genomes remain poorly annotated, due to their sequence diversity and changeable domain architecture. In order to achieve an automated annotation of these proteins that is both detailed and accurate we have taken a domain dictionary approach, in which we identify recurrent domains by sequence comparisons, produce bioinformatic descriptors for each domain type, and connect these to structural information where available. We implemented this approach in a web-based platform, daTAA, in 2008 and demonstrated its applicability by reconstructing the complete fiber structure of a TAA conserved in enterobacteria. Here we review current knowledge on the domain structure of TAAs.