Ahmad Jomaa

Role of the PDZ Domains in Escherichia coli DegP Protein

PDZ domains are modular protein interaction domains that are present in metazoans and bacteria. These domains possess unique structural features that allow them to interact with the C-terminal residues of their ligands. The Escherichia coli essential periplasmic protein DegP contains two PDZ domains attached to the C-terminal end of the protease domain. In this study we examined the role of each PDZ domain in the protease and chaperone activities of this protein. Specifically, DegP mutants with either one or both PDZ domains deleted were generated and tested to determine their protease and chaperone activities, as well as their abilities to sequester unfolded substrates. We found that the PDZ domains in DegP have different roles; the PDZ1 domain is essential for protease activity and is responsible for recognizing and sequestering unfolded substrates through C-terminal tags, whereas the PDZ2 domain is mostly involved in maintaining the hexameric cage of DegP. Interestingly, neither of the PDZ domains was required for the chaperone activity of DegP. In addition, we found that the loops connecting the protease domain to PDZ1 and connecting PDZ1 to PDZ2 are also essential for the protease activity of the hexameric DegP protein. New insights into the roles of the PDZ domains in the structure and function of DegP are provided. These results imply that DegP recognizes substrate molecules targeted for degradation and substrate molecules targeted for refolding in different manners and suggest that the substrate recognition mechanisms may play a role in the protease-chaperone switch, dictating whether the substrate is degraded or refolded.

The inner cavity of Escherichia coli DegP protein is not essential for molecular chaperone and proteolytic activity

The Escherichia coli DegP protein is an essential periplasmic protein for bacterial survival at high temperatures. DegP has the unusual property of working as a chaperone below 28 degrees C, but efficiently degrading unfolded proteins above 28 degrees C. Monomeric DegP contains a protease domain and two PDZ domains. It oligomerizes into a hexameric cage through the staggered association of trimers. The active sites are located in a central cavity that is only accessible laterally, and the 12 PDZ domains act as mobile sidewalls that mediate opening and closing of the gates. As access to the active sites is restricted, DegP is an example of a self-compartmentalized protease. To determine the essential elements of DegP that maintain the integrity of the hexameric cage, we constructed several deletion mutants of DegP that formed trimers rather than hexamers. We found that residues 39 to 78 within the LA loops, as well as the PDZ2 domains are essential for the integrity of the DegP hexamer. In addition, we asked whether an enclosed cavity or cage of specific dimensions is required for the protease and chaperone activities in DegP. Both activities were maintained in the trimeric DegP mutants without an enclosed cavity and in deletion DegP mutants with significantly reduced dimensions of the cage. We conclude that the functional unit for the protease and chaperone activities of DegP is a trimer and that neither a cavity of specific dimensions nor the presence of an enclosed cavity appears to be essential for the protease and chaperone activities of DegP.

Dual amyloid domains promote differential functioning of the chaplin proteins during Streptomyces aerial morphogenesis.

The chaplin proteins are functional amyloids found in the filamentous Streptomyces bacteria. These secreted proteins are required for the aerial development of Streptomyces coelicolor, and contribute to an intricate rodlet ultrastructure that decorates the surfaces of aerial hyphae and spores. S. coelicolor encodes eight chaplin proteins. Previous studies have revealed that only three of these proteins (ChpC, ChpE, and ChpH) are necessary for promoting aerial development, and of these three, ChpH is the primary developmental determinant. Here, we show that the model chaplin, ChpH, contains two amyloidogenic domains: one in the N terminus and one in the C terminus of the mature protein. These domains have different polymerization properties as determined using fluorescence spectroscopy, secondary structure analyses, and electron microscopy. We coupled these in vitro assays with in vivo genetic studies to probe the connection between ChpH amyloidogenesis and its biological function. Using mutational analyses, we demonstrated that both N- and C-terminal amyloid domains of ChpH were required for promoting aerial hypha formation, while the N-terminal domain was dispensable for assembly of the rodlet ultrastructure. These results suggest that there is a functional differentiation of the dual amyloid domains in the chaplin proteins.

Structures of the E. coli translating ribosome with SRP and its receptor and with the translocon.

Co-translational protein targeting to membranes is a universally conserved process. Central steps include cargo recognition by the signal recognition particle and handover to the Sec translocon. Here we present snapshots of key co-translational-targeting complexes solved by cryo-electron microscopy at near-atomic resolution, establishing the molecular contacts between the Escherichia coli translating ribosome, the signal recognition particle and the translocon. Our results reveal the conformational changes that regulate the latching of the signal sequence, the release of the heterodimeric domains of the signal recognition particle and its receptor, and the handover of the signal sequence to the translocon. We also observe that the signal recognition particle and the translocon insert-specific structural elements into the ribosomal tunnel to remodel it, possibly to sense nascent chains. Our work provides structural evidence for a conformational state of the signal recognition particle and its receptor primed for translocon binding to the ribosome–nascent chain complex.

Pch2 is a hexameric ring ATPase that remodels the chromosome axis protein Hop1

Significance The conserved PCH2 gene in baker’s yeast regulates meiotic double-strand break repair outcomes, helps establish a proper meiotic chromosome structure, and is important for the progression of meiotic recombination. Its mouse homolog is required for fertility. However, the molecular mechanism of how PCH2 regulates these diverse functions is not known. In this study, we show that Pch2 is an AAA+ family ATPase (ATPases associated with diverse cellular activities) that oligomerizes into single hexameric rings. In the presence of ATP, Pch2 binds to and remodels Hop1, an important component of the synaptonemal complex, and displaces it from DNA. Based on these and previous observations, we suggest that Pch2 impacts meiotic chromosome organization by directly regulating Hop1 binding to DNA. In budding yeast the pachytene checkpoint 2 (Pch2) protein regulates meiotic chromosome axis structure by maintaining the domain-like organization of the synaptonemal complex proteins homolog pairing 1 (Hop1) and molecular zipper 1 (Zip1). Pch2 has also been shown to modulate meiotic double-strand break repair outcomes to favor recombination between homologs, play an important role in the progression of meiotic recombination, and maintain ribosomal DNA stability. Pch2 homologs are present in fruit flies, worms, and mammals, however the molecular mechanism of Pch2 function is unknown. In this study we provide a unique and detailed biochemical analysis of Pch2. We find that purified Pch2 is an AAA+ (ATPases associated with diverse cellular activities) protein that oligomerizes into single hexameric rings in the presence of nucleotides. In addition, we show Pch2 binds to Hop1, a critical axial component of the synaptonemal complex that establishes interhomolog repair bias, in a nucleotide-dependent fashion. Importantly, we demonstrate that Pch2 displaces Hop1 from large DNA substrates and that both ATP binding and hydrolysis by Pch2 are required for Pch2–Hop1 transactions. Based on these and previous cell biological observations, we suggest that Pch2 impacts meiotic chromosome function by directly regulating Hop1 localization.

Functional domains of the 50S subunit mature late in the assembly process

Despite the identification of many factors that facilitate ribosome assembly, the molecular mechanisms by which they drive ribosome biogenesis are poorly understood. Here, we analyze the late stages of assembly of the 50S subunit using Bacillus subtilis cells depleted of RbgA, a highly conserved GTPase. We found that RbgA-depleted cells accumulate late assembly intermediates bearing sub-stoichiometric quantities of ribosomal proteins L16, L27, L28, L33a, L35 and L36. Using a novel pulse labeling/quantitative mass spectrometry technique, we show that this particle is physiologically relevant and is capable of maturing into a complete 50S particle. Cryo-electron microscopy and chemical probing revealed that the central protuberance, the GTPase associating region and tRNA-binding sites in this intermediate are unstructured. These findings demonstrate that key functional sites of the 50S subunit remain unstructured until late stages of maturation, preventing the incomplete subunit from prematurely engaging in translation. Finally, structural and biochemical analysis of a ribosome particle depleted of L16 indicate that L16 binding is necessary for the stimulation of RbgA GTPase activity and, in turn, release of this co-factor, and for conversion of the intermediate to a complete 50S subunit.

Characterization of the Autocleavage Process of the Escherichia coli HtrA Protein: Implications for its Physiological Role

The Escherichia coli HtrA protein is a periplasmic protease/chaperone that is upregulated under stress conditions. The protease and chaperone activities of HtrA eliminate or refold damaged and unfolded proteins in the bacterial periplasm that are generated upon stress conditions. In the absence of substrates, HtrA oligomerizes into a hexameric cage, but binding of misfolded proteins transforms the hexamers into bigger 12-mer and 24-mer cages that encapsulate the substrates for degradation or refolding. HtrA also undergoes partial degradation as a consequence of self-cleavage of the mature protein, producing short-HtrA protein (s-HtrA). The aim of this study was to examine the physiological role of this self-cleavage process. We found that the only requirement for self-cleavage of HtrA into s-HtrA in vitro was the hydrolysis of protein substrates. In fact, peptides resulting from the hydrolysis of the protein substrates were sufficient to induce autocleavage. However, the continuous presence of full-length substrate delayed the process. In addition, we observed that the hexameric cage structure is required for autocleavage and that s-HtrA accumulates only late in the degradation reaction. These results suggest that self-cleavage occurs when HtrA reassembles back into the resting hexameric structure and peptides resulting from substrate hydrolysis are allosterically stimulating the HtrA proteolytic activity. Our data support a model in which the physiological role of the self-cleavage process is to eliminate the excess of HtrA once the stress conditions cease.

Escherichia coli DegP: a Structure-Driven Functional Model

Structural biology has been extremely successful in providing functional insights for a large number of proteins and macromolecular assemblies, but in some cases, the structure has contributed a three-dimensional (3D) framework to interpret years of accumulated biochemical and genetic knowledge. In these particular systems, structural information has allowed us to learn things that would have been difficult to learn with other techniques. The periplasmic Escherichia coli DegP protease/chaperone exemplifies this scenario very well. This protein was initially identified in the 1980s (23, 24, 37, 38), and over more than two decades, several groups characterized its activities (2, 12, 16, 27). However, a comprehensive 3D functional model did not become apparent until the first DegP X-ray structure was revealed in 2002 (20). Following the discovery of this remarkable structure, a number of groups concentrated on testing the essentials of the functional model proposed from the crystal structure. Interestingly, this functional model was rewritten recently, after the structures of DegP in two additional oligomeric forms were resolved (13, 22). Current research efforts are now concentrated on probing the new functional model and also on answering new, interesting questions posed by these structures. Therefore, DegP provides an excellent example for the structure-driven study of protein function. This minireview aims to summarize how the functional model for DegP protein has evolved as the structures of the different oligomeric forms of the protein have been elucidated. E. coli DegP (also called HtrA or protease Do) is an important periplasmic protein with the unusual property of functioning both as a protease and as a chaperone (36). Unlike the cytoplasmic compartment, the periplasm lacks ATP and does not support the function of large protein machines powered by this molecule. However, in this cellular compartment, DegP can still degrade and refold misfolded proteins in an ATPindependent manner (2). Although DegP is not an essential protein, its activity is required for bacterial survival at high temperatures (34) and under harsh environmental conditions. Consequently, its expression is upregulated by both the Cpx and E protein quality control pathways under conditions of protein-folding stress (3, 29). DegP homologs have been isolated from a variety of species, including gram-negative and -positive bacteria, plants, and mammals. All these proteins constitute the HtrA family of proteases (2). In bacteria, members of this family are key players mainly in protein quality control in the periplasmic space. In eukaryotic cells, these proteins are involved in functions as diverse as the regulation of apoptosis (1, 4, 9) and the delay of the aggregation process of intracellular amyloid peptides (19). HtrA proteins usually contain a protease domain and at least one C-terminal PDZ domain. In some cases, members of this family of proteins also include additional domains, such as transmembrane regions, located usually at the N terminus. Specifically, in E. coli, DegP, DegQ, and DegS compose the HtrA family. There is a high degree of homology among these three proteins, particularly in the protease domain. However, the number of PDZ domains is variable. DegP and DegQ contain two PDZ domains, whereas DegS contains only one (16).