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Abstract

The Clp/Hsp100 ATPases are hexameric protein machines that catalyze the unfolding, disassembly and disaggregation of specific protein substrates in bacteria, plants and animals. Many family members also interact with peptidases to form ATP-dependent proteases. In Escherichia coli, for instance, the ClpXP protease is assembled from the ClpX ATPase and the ClpP peptidase. Here, we have used multiple sequence alignments to identify a tripeptide 'IGF' in E. coli ClpX that is essential for ClpP recognition. Mutations in this IGF sequence, which appears to be part of a surface loop, disrupt ClpXP complex formation and prevent protease function but have no effect on other ClpX activities. Homologous tripeptides are found only in a subset of Clp/Hsp100 ATPases and are a good predictor of family members that have a ClpP partner. Mapping of the IGF loop onto a homolog of known structure suggests a model for ClpX-ClpP docking.

Crystal Structure of ClpX Molecular Chaperone from Helicobacter pylori

“…As the amino acid sequence of the tripeptide is relatively well conserved among the ClpP binding loops and neighboring helices located between the two conserved motifs, sensor I and box VII (12), the LGF peptide might be critical for the initial recognition of ClpP, and the other loops and helices (␣8 -␣10) could act as linkers or be involved in nonspecific binding to the ClpP. These structural interpretations are in agreement with proteolytic (10) and mutation experiments (13), which proposed that the tripeptide is open to the protease interface surface, and involved in ClpP recognition. The C-terminal residues of HslU, which are missing in ClpX, are thought to take part in protease recognition.…”

“…Considering that 8 amino acids in the C terminus of Hp ClpX are invisible in the current structure, the C-terminal end could be extending from the core of SSD domain toward the substrate interface. Unlike HslU, the Clp family is expected to have a ClpP binding loop, with the conserved tripeptide (LIV)-G-(FL) in the loop suggested to be a candidate for the ClpP recognition motif (13). Hp ClpX contains the ClpP binding loop, with additional surrounding helices (␣8, ␣9, and ␣10), which are absent in E. coli HslU (Fig.…”

“…The crystal structures of HslU(ClpY) complexed with HslV(ClpQ) revealed that it contains one ATPase domain with similar structural architecture to ClpA D2 (18). Despite the structural resemblance of the ATPase domains, it is expected that HslU has a different structural feature, as it binds to HslV hexamer through the C-terminal loop, while ClpA or ClpX binds to the ClpP heptamer through the ClpP binding loop (13).…”

“…There is an additional small Cys cluster domain at the N terminus. The ATPase core domain of ClpX contains a tripeptide (LIV)-G-(FL) that is responsible for ClpP recognition and unique to ClpA and ClpX (13). The C-terminal SSD domain is known to be involved in substrate binding specificity (14).…”

NblA, a Key Protein of Phycobilisome Degradation, Interacts with ClpC, a HSP100 Chaperone Partner of a Cyanobacterial Clp Protease

“…Using sequence analysis we ascertained that SyClpC is the ortholog of Alr2999 from Nostoc 7120 (ClpC Alr2999 ), the here identified interaction partner of NblA. Both proteins show a domain composition typical for ClpC proteins and possess the (L/I)GF motif responsible for the association with the Clp core (37,38). In conclusion, our findings are underscored by the results reported by Stanne and co-workers (41) and lead to the proposal that PBS degradation is caused by NblA binding to ClpC, the regulatory subunit of a membraneassociated Clp protease.…”