scholarly journals Structural basis of ClpXP recognition and unfolding of ssrA-tagged substrates

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Xue Fei ◽  
Tristan A Bell ◽  
Sarah R Barkow ◽  
Tania A Baker ◽  
Robert T Sauer
Keyword(s):  
Escherichia Coli ◽  
Open Channel ◽  
Structural Basis ◽  
Closed Channel ◽  
Protein Substrates ◽  
Specific Recognition ◽  
Axial Channel ◽  
Engagement Strategies ◽  
C Terminus ◽  
Protein Remodeling

When ribosomes fail to complete normal translation, all cells have mechanisms to ensure degradation of the resulting partial proteins to safeguard proteome integrity. In Escherichia coli and other eubacteria, the tmRNA system rescues stalled ribosomes and adds an ssrA tag or degron to the C-terminus of the incomplete protein, which directs degradation by the AAA+ ClpXP protease. Here, we present cryo-EM structures of ClpXP bound to the ssrA degron. C-terminal residues of the ssrA degron initially bind in the top of an otherwise closed ClpX axial channel and subsequently move deeper into an open channel. For short-degron protein substrates, we show that unfolding can occur directly from the initial closed-channel complex. For longer degron substrates, our studies illuminate how ClpXP transitions from specific recognition into a nonspecific unfolding and translocation machine. Many AAA+ proteases and protein-remodeling motors are likely to employ similar multistep recognition and engagement strategies.

scholarly journals Structural basis of ClpXP recognition and unfolding of ssrA-tagged substrates

2020 ◽  
Author(s):  
Xue Fei ◽  
Tristan A Bell ◽  
Sarah R Barkow ◽  
Tania A Baker ◽  
Robert T Sauer
Keyword(s):  
Open Channel ◽  
Structural Basis ◽  
Closed Channel ◽  
E Coli ◽  
Protein Substrates ◽  
Specific Recognition ◽  
Axial Channel ◽  
Engagement Strategies ◽  
C Terminus ◽  
Protein Remodeling

ABSTRACTWhen ribosomes fail to complete normal translation, all cells have mechanisms to ensure degradation of the resulting partial proteins to safeguard proteome integrity. In E. coli and other eubacteria, the tmRNA system rescues stalled ribosomes and adds an ssrA tag or degron to the C-terminus of the incomplete protein, which directs degradation by the AAA+ ClpXP protease. Here, we present cryo-EM structures of ClpXP bound to the ssrA degron. C-terminal residues of the ssrA degron initially bind in the top of an otherwise closed ClpX axial channel and subsequently move deeper into an open channel. For short-degron protein substrates, we show that unfolding can occur directly from the initial closed-channel complex. For longer-degron substrates, our studies illuminate how ClpXP transitions from specific recognition into a nonspecific unfolding and translocation machine. Many AAA+ proteases and protein-remodeling motors are likely to employ similar multistep recognition and engagement strategies.

scholarly journals Clusters of Charged Residues at the C Terminus of MotA and N Terminus of MotB Are Important for Function of the Escherichia coli Flagellar Motor

2008 ◽  
Vol 190 (15) ◽  
pp. 5517-5521 ◽  
Author(s):  
Edan R. Hosking ◽  
Michael D. Manson
Keyword(s):  
Escherichia Coli ◽  
Flagellar Motor ◽  
Protein Levels ◽  
Partner Protein ◽  
C Terminus ◽  
N Terminus ◽  
Charged Residues ◽  
Positively Charged ◽  
Terminal Cluster

ABSTRACT MotA contains a conserved C-terminal cluster of negatively charged residues, and MotB contains a conserved N-terminal cluster of positively charged residues. Charge-altering mutations affecting these residues impair motility but do not diminish Mot protein levels. The motility defects are reversed by second-site mutations targeting the same or partner protein.

scholarly journals Structure of undecaprenyl pyrophosphate synthase from Acinetobacter baumannii

2018 ◽  
Vol 74 (12) ◽  
pp. 765-769 ◽  
Author(s):  
Tzu-Ping Ko ◽  
Chi-Hung Huang ◽  
Shu-Jung Lai ◽  
Yeh Chen
Keyword(s):  
Acinetobacter Baumannii ◽  
Active Site ◽  
Multidrug Resistant ◽  
Structural Basis ◽  
X Ray Diffraction ◽  
X Ray ◽  
Peptidoglycan Synthesis ◽  
C Terminus ◽  
Dimeric Protein ◽  
Β Sheet

Undecaprenyl pyrophosphate (UPP) is an important carrier of the oligosaccharide component in peptidoglycan synthesis. Inhibition of UPP synthase (UPPS) may be an effective strategy in combating the pathogen Acinetobacter baumannii, which has evolved to be multidrug-resistant. Here, A. baumannii UPPS (AbUPPS) was cloned, expressed, purified and crystallized, and its structure was determined by X-ray diffraction. Each chain of the dimeric protein folds into a central β-sheet with several surrounding α-helices, including one at the C-terminus. In the active site, two molecules of citrate interact with the side chains of the catalytic aspartate and serine. These observations may provide a structural basis for inhibitor design against AbUPPS.

scholarly journals Structural Basis for the Specific Recognition of RET by the Dok1 Phosphotyrosine Binding Domain

2003 ◽  
Vol 279 (6) ◽  
pp. 4962-4969 ◽  
Author(s):  
Ning Shi ◽  
Sheng Ye ◽  
Mark Bartlam ◽  
Maojun Yang ◽  
Jing Wu ◽  
...  
Keyword(s):  
Binding Domain ◽  
Structural Basis ◽  
Specific Recognition ◽  
Phosphotyrosine Binding Domain

scholarly journals Yeast-Based Directed-Evolution For High-Throughput Structural Stabilization of G Protein-Coupled Receptors (GPCRs)

2021 ◽  
Author(s):  
May Meltzer ◽  
Zvagelsky Tatiana ◽  
Niv Papo ◽  
Stanislav Engel
Keyword(s):  
G Protein ◽  
Resistant Cell ◽  
Single Step ◽  
G Protein Coupled Receptors ◽  
Structural Basis ◽  
Diffusion Method ◽  
Structural Stabilization ◽  
C Terminus ◽  
Screening Platform ◽  
G Protein Coupled

Abstract The immense potential of G protein-coupled receptors (GPCRs) as targets for drug discovery is not fully realized due to the enormous difficulties associated with structure elucidation of these profoundly unstable membrane proteins. The existing methods of GPCR stability-engineering are cumbersome and low-throughput; in addition, the scope of GPCRs that could benefit from these techniques is limited. Here, we presented a yeast-based screening platform for a single-step isolation of GRCR variants stable in the presence of short-chain detergents, a feature essential for their successful crystallization using vapor diffusion method. The detergent-resistant cell wall of yeast provides a unique compartmentalization opportunity to physically link the receptor phenotype to its encoding DNA, and thus enable discovery of stable GPCR variants with unprecedent efficiency. The scope of mutations identified by the method offers important insights into the structural basis of GPCR stability, questioning the inherent instability of the GPCR scaffold, and revealing the potential role of the C-terminus in receptor stabilization.

scholarly journals Structural basis for UFM1 transfer from UBA5 to UFC1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Manoj Kumar ◽  
Prasanth Padala ◽  
Jamal Fahoum ◽  
Fouad Hassouna ◽  
Tomer Tsaban ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Structural Basis ◽  
Adenylation Domain ◽  
Post Translational Modification ◽  
Target Proteins ◽  
Cellular Processes ◽  
Enzyme Cascade ◽  
Linear Sequence ◽  
C Terminus

AbstractUfmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1’s active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1’s conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.

scholarly journals The C-Terminal Flexible Domain of the Heme Chaperone CcmE Is Important but Not Essential for Its Function

2003 ◽  
Vol 185 (13) ◽  
pp. 3821-3827 ◽  
Author(s):  
Elisabeth Enggist ◽  
Linda Thöny-Meyer
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Heme Binding ◽  
Membrane Anchor ◽  
Terminal Domain ◽  
Conserved Domain ◽  
C Terminus ◽  
Helical Turn ◽  
Low Activity

ABSTRACT CcmE is a heme chaperone active in the cytochrome c maturation pathway of Escherichia coli. It first binds heme covalently to strictly conserved histidine H130 and subsequently delivers it to apo-cytochrome c. The recently solved structure of soluble CcmE revealed a compact core consisting of a β-barrel and a flexible C-terminal domain with a short α-helical turn. In order to elucidate the function of this poorly conserved domain, CcmE was truncated stepwise from the C terminus. Removal of all 29 amino acids up to crucial histidine 130 did not abolish heme binding completely. For detectable transfer of heme to type c cytochromes, only one additional residue, D131, was required, and for efficient cytochrome c maturation, the seven-residue sequence 131DENYTPP137 was required. When soluble forms of CcmE were expressed in the periplasm, the C-terminal domain had to be slightly longer to allow detection of holo-CcmE. Soluble full-length CcmE had low activity in cytochrome c maturation, indicating the importance of the N-terminal membrane anchor for the in vivo function of CcmE.

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