Analysis of the ATP-independent chaperone activity using DegQ from E. coli

Alle Zellen nutzen Proteasen und Chaperone um ein kompetentes Qualitätskontrolle-System aufzubauen, das die Akkumulierung von ungefaltenen Proteinen verhindert. In der Zellhülle von Escherichia coli wird die Qualität von Proteinen durch die HtrA-Proteasen DegP, DegS und DegQ kontrolliert. Das Ziel dieses Projekts ist es, die molekularen Funktionen von DegQ zu bestimmen, das gleichzeitig eine Protease und ein ATP-unabhängiges Chaperon im bakteriellen Periplasma darstellt. Es zeigte sich, dass die Umwandlung von ruhenden DegQ Hexameren in katalytisch aktive 12- und 24- mere Partikel durch die Bindung von Substraten ausgelöst wird. Interessanterweise hängt diese Substrat-induzierte Umwandlung des Oligomers und die Aktivierung der Proteaseaktivitat von der Anwesenheit der ersten PDZ Domäne ab, jedoch nicht von der zweiten. Diese Beobachtung impliziert, dass der beschriebene Regulationsmechanismus ein allgemeines Merkmal der HtrAProteasen ist, die üblicherweise nur eine PDZ Domäne besitzen. Unsere in vitro und in vivo Daten zeigen des Weiteren, dass die Funktion von DegQ in der bakteriellen Zellhülle vom pH-Wert bestimmt wird. Eine DegQ Mutante, der die zweite PDZ Domäne fehlt wurde für strukturbiologische Studien benutzt und die hoch-auflösende Kristallstruktur eines dodekameren HtrA-Komplexes wurde bestimmt. Die Struktur offenbarte eine konservierte Signalkaskade in der Substratbindung und Proteaseaktivierung gekoppelt sind. Weitere elektronenmikroskopische Studien von Volllängen-DegQ deuteten darauf hin, dass DegQ zusätzlich zur Proteaseaktivität auch Chaperonaktivität besitzt. Um die molekularen Aspekte der Chaperonaktivität zu charakterisieren wurde eine Mutationsanalyse durchgeführt. Die DegQ Mutanten wurden basierend auf der funktionellen Analogie zu Chaperoninen konstruiert, welche ihre exponierten hydrophoben Bindungsstellen zur Substratbindung nutzen und nach einer Konformationsänderung eine hydrophile Kammer zur Proteinfaltung ausbilden. DegQ hingegegen bietet hydrophobe Bindungsstellen in der ersten PDZ Domäne in seinem hexameren Zustand und formt eine geschlossene hydrophile Umgebung in seiner dodekameren Form. Eine detaillierte biochemische Charakterisierung der DegQ Mutanten zeigte, dass die Mutation der Reste F257 und F266 in der ersten PDZ Domäne in der Tat die Chaperonaktivität beeinflusst. Es sind jedoch noch weitere Experimente nötig, um die zugrundeliegenden Ursachen dieser Beeinträchtigung herauszufinden. Des Weiteren zeigte diese Studie, dass die Mutationen die Proteaseaktivität in einer Substrat-spezifischen Weise beeinflusst haben und entweder zu niedrigeren Abbauraten oder unterschiedlichen Abbauprodukten geführt haben. Insgesamt bieten die durchgeführten Studien wichtige Einblicke in die Struktur und Funktion von DegQ und tragen zu einem besseren Verständnis der zugrundeliegenen allgemeinen Mechanismen der Protease- und Chaperonaktiviät von HtrAProteinen bei.All cells employ proteases and chaperones to setup a competent quality control system, preventing the accumulation of misfolded proteins. In Escherichia coli, the quality of the proteins in the cell envelope is controlled by the HtrA proteases DegP, DegS and DegQ. The aim of the project is to determine the molecular features of DegQ, which represents a protease and concomitantly an ATP-independent chaperone residing in the bacterial periplasm. Our studies showed that substrate binding triggers the conversion of the resting DegQ hexamer into catalytically active 12- and 24-mers. Interestingly, substrate-induced oligomer reassembly and protease activation depends on the first PDZ domain, but not on the second. This result implies that the mentioned regulatory mechanism may be a common feature of HtrA proteases that typically encompass a single PDZ domain. Furthermore, our in vitro and in vivo data point to a pH-related function of DegQ in the bacterial cell envelope. In addition, a DegQ mutant lacking the second PDZ domain was used for structural studies and the high-resolution crystal structure of a dodecameric HtrA complex was determined. The obtained structural data revealed a conserved signaling cascade in which substrate binding and protease activation are coupled. Further structural studies of full length DegQ, using Electron Microscopy, suggested that in addition to protease activity, DegQ has also chaperone activity. In order to characterize the molecular aspects of the chaperone function of DegQ, a mutational analysis was performed. DegQ mutants were designed based on the functional analogy to chaperonins which expose their hydrophobic binding sites to interact with substrates and upon a conformational rearrangement provide a hydrophilic chamber to promote protein folding. DegQ in turn exposes hydrophobic binding sites within its PDZ 1 domain in the hexameric state and provides an enclosed hydrophilic environment upon dodecamer formation. A detailed biochemical characterization of DegQ mutants revealed that mutations of residues F257 and F266 in the PDZ 1 domain indeed affected the chaperone activity of DegQ. However, further experiments are required to elucidate the underlying causes of the impairment in detail. Additionally, the study showed that the protease activity is affected by the mutations in a substrate-specific manner leading to either lower degradation rates or different degradation products. Taken together, the performed studies provide important insights in the DegQ structure and function leading to a better understanding of general mechanisms underlying the protease and chaperone activities of HtrA proteins

Molecular adaptation of the DegQ protease to exert protein quality control in the bacterial cell envelope

To react to distinct stress situations and to prevent the accumulation of misfolded proteins, all cells employ a number of proteases and chaperones, which together set up an efficient protein quality control system. The functionality of proteins in the cell envelope of Escherichia coli is monitored by the HtrA proteases DegS, DegP, and DegQ. In contrast with DegP and DegS, the structure and function of DegQ has not been addressed in detail. Here, we show that substrate binding triggers the conversion of the resting DegQ hexamer into catalytically active 12- and 24-mers. Interestingly, substrate-induced oligomer reassembly and protease activation depends on the first PDZ domain but not on the second. Therefore, the regulatory mechanism originally identified in DegP should be a common feature of HtrA proteases, most of which encompass only a single PDZ domain. Using a DegQ mutant lacking the second PDZ domain, we determined the high resolution crystal structure of a dodecameric HtrA complex. The nearly identical domain orientation of protease and PDZ domains within 12- and 24-meric HtrA complexes reveals a conserved PDZ1 → L3 → LD/L1/L2 signaling cascade, in which loop L3 senses the repositioned PDZ1 domain of higher order, substrate-engaged particles and activates protease function. Furthermore, our in vitro and in vivo data imply a pH-related function of DegQ in the bacterial cell envelope

Substrate-induced remodeling of the active site regulates human HTRA1 activity

Crystal structures of active and inactive conformations of the human serine protease HTRA1 reveal that substrate binding to the active site is sufficient to stimulate proteolytic activity. HTRA1 attaches to liposomes, digests misfolded proteins into defined fragments and undergoes substrate-mediated oligomer conversion. In contrast to those of other serine proteases, the PDZ domain of HTRA1 is dispensable for activation or lipid attachment, indicative of different underlying mechanistic features

Molecular Adaptation of the DegQ Protease to Exert Protein Quality Control in the Bacterial Cell Envelope*

To react to distinct stress situations and to prevent the accumulation of misfolded proteins, all cells employ a number of proteases and chaperones, which together set up an efficient protein quality control system. The functionality of proteins in the cell envelope of Escherichia coli is monitored by the HtrA proteases DegS, DegP, and DegQ. In contrast with DegP and DegS, the structure and function of DegQ has not been addressed in detail. Here, we show that substrate binding triggers the conversion of the resting DegQ hexamer into catalytically active 12- and 24-mers. Interestingly, substrate-induced oligomer reassembly and protease activation depends on the first PDZ domain but not on the second. Therefore, the regulatory mechanism originally identified in DegP should be a common feature of HtrA proteases, most of which encompass only a single PDZ domain. Using a DegQ mutant lacking the second PDZ domain, we determined the high resolution crystal structure of a dodecameric HtrA complex. The nearly identical domain orientation of protease and PDZ domains within 12- and 24-meric HtrA complexes reveals a conserved PDZ1 → L3 → LD/L1/L2 signaling cascade, in which loop L3 senses the repositioned PDZ1 domain of higher order, substrate-engaged particles and activates protease function. Furthermore, our in vitro and in vivo data imply a pH-related function of DegQ in the bacterial cell envelope

Newly folded substrates inside the molecular cage of the HtrA chaperone DegQ

The HtrA protein family combines chaperone and protease activities and is essential for protein quality control in many organisms. Whereas the mechanisms underlying the proteolytic function of HtrA proteins are well characterized, their chaperone activity remains poorly understood. Here we describe cryo-EM structures of Escherichia coli DegQ in its 12- and 24-mer states in complex with model substrates, providing a structural model of HtrA chaperone action. Up to six lysozyme substrates bind inside the DegQ 12-mer cage and are visualized in a close-to-native state. An asymmetric reconstruction reveals the binding of a well-ordered lysozyme to four DegQ protomers. DegQ PDZ domains are located adjacent to substrate density and their presence is required for chaperone activity. The substrate-interacting regions appear conserved in 12- and 24-mer cages, suggesting a common mechanism of chaperone function