Molecular cloning of an actinobacterial-type ClpS gene from Celosia cristata expression library

In proteobacterial cytosol, ClpS protein is known as a molecular adaptor for substrate selectivity and proteolytic activity of the ATP-dependent chaperone-protease complex, ClpAP. ClpA-related ClpS is a small protein usually encoded immediately upstream of ClpA in the genome of proteobacteria. Recent bioinformatics analysis has revealed the presence of cyanobacterial-type ClpS or ClpC-related ClpS in organisms lacking ClpA, including all the plant species sequenced to date. Here we report the identification of an actinobacterial homologue of the ClpS (possibly Clp-related) gene from a plant system. A cDNA, spanning 566 bp with a complete coding region corresponding to 132 amino acids, was isolated from a Celosia cristata expression library constructed on a λ TriplEX2 vector. This cDNA product was considered to be an ATP-dependent Clp protease adaptor and was designated as Celosia actinobacterial-type ClpS, since it contains a highly conserved domain belonging to the ClpS family of proteins from actinobacteria. Celosia ClpS is about 80% identical to actinobacterial ClpS proteins in its overall deduced amino acid sequence. Based on this finding, we may define a novel target of ATP-dependent Clp complex in a plant system or speculate the presence of a second type of molecular chaperone besides ClpC in plants, as predicted for actinobacteria.     

Structural basis for the N‐degron specificity of ClpS1 from Arabidopsis thaliana

The N‐degron pathway determines the half‐life of proteins in both prokaryotes and eukaryotes by precisely recognizing the N‐terminal residue (N‐degron) of substrates. ClpS proteins from bacteria bind to substrates containing hydrophobic N‐degrons (Leu, Phe, Tyr, and Trp) and deliver them to the caseinolytic protease system ClpAP. This mechanism is preserved in organelles such as mitochondria and chloroplasts. Bacterial ClpS adaptors bind preferentially to Leu and Phe N‐degrons; however, ClpS1 from Arabidopsis thaliana (AtClpS1) shows a difference in that it binds strongly to Phe and Trp N‐degrons and only weakly to Leu. This difference in behavior cannot be explained without structural information due to the high sequence homology between bacterial and plant ClpS proteins. Here, we report the structure of AtClpS1 at 2.0 Å resolution in the presence of a bound N‐degron. The key determinants for α‐amino group recognition are conserved among all ClpS proteins, but the α3‐helix of eukaryotic AtClpS1 is significantly shortened, and consequently, a loop forming a pocket for the N‐degron is moved slightly outward to enlarge the pocket. In addition, amino acid replacement from Val to Ala causes a reduction in hydrophobic interactions with Leu N‐degron. A combination of the fine‐tuned hydrophobic residues in the pocket and the basic gatekeeper at the entrance of the pocket controls the N‐degron selectivity of the plant ClpS protein.     

Structure of a putative ClpS N‐end rule adaptor protein from the malaria pathogen Plasmodium falciparum

The N‐end rule pathway uses an evolutionarily conserved mechanism in bacteria and eukaryotes that marks proteins for degradation by ATP‐dependent chaperones and proteases such as the Clp chaperones and proteases. Specific N‐terminal amino acids (N‐degrons) are sufficient to target substrates for degradation. In bacteria, the ClpS adaptor binds and delivers N‐end rule substrates for their degradation upon association with the ClpA/P chaperone/protease. Here, we report the first crystal structure, solved at 2.7 Å resolution, of a eukaryotic homolog of bacterial ClpS from the malaria apicomplexan parasite Plasmodium falciparum (Pfal). Despite limited sequence identity, Plasmodium ClpS is very similar to bacterial ClpS. Akin to its bacterial orthologs, plasmodial ClpS harbors a preformed hydrophobic pocket whose geometry and chemical properties are compatible with the binding of N‐degrons. However, while the N‐degron binding pocket in bacterial ClpS structures is open and accessible, the corresponding pocket in Plasmodium ClpS is occluded by a conserved surface loop that acts as a latch. Despite the closed conformation observed in the crystal, we show that, in solution, Pfal‐ClpS binds and discriminates peptides mimicking bona fide N‐end rule substrates. The presence of an apicoplast targeting peptide suggests that Pfal‐ClpS localizes to this plastid‐like organelle characteristic of all Apicomplexa and hosting most of its Clp machinery. By analogy with the related ClpS1 from plant chloroplasts and cyanobacteria, Plasmodium ClpS likely functions in association with ClpC in the apicoplast. Our findings open new venues for the design of novel anti‐malarial drugs aimed at disrupting parasite‐specific protein quality control pathways.     

Modification of PATase by L/F‐transferase generates a ClpS‐dependent N‐end rule substrate in Escherichia coli

The N‐end rule pathway is conserved from bacteria to man and determines the half‐life of a protein based on its N‐terminal amino acid. In Escherichia coli, model substrates bearing an N‐degron are recognised by ClpS and degraded by ClpAP in an ATP‐dependent manner. Here, we report the isolation of 23 ClpS‐interacting proteins from E. coli. Our data show that at least one of these interacting proteins—putrescine aminotransferase (PATase)—is post‐translationally modified to generate a primary N‐degron. Remarkably, the N‐terminal modification of PATase is generated by a new specificity of leucyl/phenylalanyl‐tRNA‐protein transferase (LFTR), in which various combinations of primary destabilising residues (Leu and Phe) are attached to the N‐terminal Met. This modification (of PATase), by LFTR, is essential not only for its recognition by ClpS, but also determines the stability of the protein in vivo. Thus, the N‐end rule pathway, through the ClpAPS‐mediated turnover of PATase may have an important function in putrescine homeostasis. In addition, we have identified a new element within the N‐degron, which is required for substrate delivery to ClpA.     

Sequence analysis of a salt tolerant metagenomic clone

Metagenome represent an unlimited resource for discovery of novel genes. Here we report, sequence analysis of a salt tolerant metagenomic clone (6B4) from a pond water metagenomic library. Clone 6B4 had an insert of 2254 bp with G+C composition of 64.06%. DNA sequence from 6B4 showed homology to DNA sequences from pro-teobacteria indicating origin of 6B4 metagenomic insert from a yet uncharacterized proteobacteria. Two encoded proteins from clone 6B4 showed match with ATP-depen-dent Clp protease adaptor protein (ClpS) and phasin, while two truncated encoded proteins showed match with poly-3-hydroxybutyrate synthase and permease. Clp complex is known to play a role in stress tolerance. Expression of ClpS from metagenomic clone is proposed to be responsible for salt tolerance of the metagenomic clone 6B4.     

Structural basis of N‐end rule substrate recognition in Escherichia coli by the ClpAP adaptor protein ClpS

In Escherichia coli, the ClpAP protease, together with the adaptor protein ClpS, is responsible for the degradation of proteins bearing an amino‐terminal destabilizing amino acid (N‐degron). Here, we determined the three‐dimensional structures of ClpS in complex with three peptides, each having a different destabilizing residue—Leu, Phe or Trp—at its N terminus. All peptides, regardless of the identity of their N‐terminal residue, are bound in a surface pocket on ClpS in a stereo‐specific manner. Several highly conserved residues in this binding pocket interact directly with the backbone of the N‐degron peptide and hence are crucial for the binding of all N‐degrons. By contrast, two hydrophobic residues define the volume of the binding pocket and influence the specificity of ClpS. Taken together, our data suggest that ClpS has been optimized for the binding and delivery of N‐degrons containing an N‐terminal Phe or Leu.     

定量蛋白质组学分析ClpS在分枝杆菌耐药中的功能

ClpS是原核生物蛋白质降解复合物ClpAPS的重要组成成分,它可以识别某些特定的氨基酸序列并将其呈递给ClpAP以促进其降解。同时,ClpS也抑制了其他蛋白质底物的降解。本研究通过在耻垢分枝杆菌中过度表达ClpS,发现所构建的重组菌株提高了利福平的抗药性。应用定量蛋白质组学技术,我们系统地分析了过度表达ClpS对于细菌蛋白质组的影响,并推测出细菌抗利福平的分子机制:ClpS促进稳态的调整、促进药物沉降以及加速药物代谢。本研究首次通过改变细菌降解复合物的相关蛋白的表达增加细菌的抗药性,并证明蛋白质组学技术是细菌的抗药性研究以及耐药株筛选的重要工具。