Robert T. Sauer

Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA.

Variants of λ repressor and cytochrome b562 translated from messenger RNAs without stop codons were modified by carboxyl terminal addition of an ssrA-encoded peptide tag and subsequently degraded by carboxyl terminal-specific proteases present in both the cytoplasm and periplasm of Escherichia coli. The tag appears to be added to the carboxyl terminus of the nascent polypeptide chain by cotranslational switching of the ribosome from the damaged messenger RNA to ssrA RNA.

Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals.

ClpXP is a protease involved in DNA damage repair, stationary-phase gene expression, and ssrA-mediated protein quality control. To date, however, only a handful of ClpXP substrates have been identified. Using a tagged and inactive variant of ClpP, substrates of E. coli ClpXP were trapped in vivo, purified, and identified by mass spectrometry. The more than 50 trapped proteins include transcription factors, metabolic enzymes, and proteins involved in the starvation and oxidative stress responses. Analysis of the sequences of the trapped proteins revealed five recurring motifs: two located at the C terminus of proteins, and three N-terminal motifs. Deletion analysis, fusion proteins, and point mutations established that sequences from each motif class targeted proteins for degradation by ClpXP. These results represent a description of general rules governing substrate recognition by a AAA+ family ATPase and suggest strategies for regulation of protein degradation.

OMP Peptide Signals Initiate the Envelope-Stress Response by Activating DegS Protease via Relief of Inhibition Mediated by Its PDZ Domain

Transmembrane signaling between intracellular compartments is often controlled by regulated proteolysis. Escherichia coli respond to misfolded or unfolded outer-membrane porins (OMPs) in the periplasm by inducing sigma(E)-dependent transcription of stress genes in the cytoplasm. This process requires a proteolytic cascade initiated by the DegS protease, which destroys a transmembrane protein (RseA) that normally binds to and inhibits sigma(E). Here, we show that peptides ending with OMP-like C-terminal sequences bind the DegS PDZ domain, activate DegS cleavage of RseA, and induce sigma(E)-dependent transcription. These results suggest that DegS acts as a sensor of envelope stress by binding unassembled OMPs. DegS activation involves relief of inhibitory interactions between its PDZ and protease domains. Peptide binding to inhibitory PDZ domains in proteases related to DegS, including DegP/HtrA, may also regulate the degradation of specific substrates by these enzymes.

AAA+ Proteases: ATP-Fueled Machines of Protein Destruction

AAA+ family proteolytic machines (ClpXP, ClpAP, ClpCP, HslUV, Lon, FtsH, PAN/20S, and the 26S proteasome) perform protein quality control and are used in regulatory circuits in all cells. These machines contain a compartmental protease, with active sites sequestered in an interior chamber, and a hexameric ring of AAA+ ATPases. Substrate proteins are tethered to the ring, either directly or via adaptor proteins. An unstructured region of the substrate is engaged in the axial pore of the AAA+ ring, and cycles of ATP binding/hydrolysis drive conformational changes that create pulses of pulling that denature the substrate and translocate the unfolded polypeptide through the pore and into the degradation chamber. Here, we review our current understanding of the molecular mechanisms of substrate recognition, adaptor function, and ATP-fueled unfolding and translocation. The unfolding activities of these and related AAA+ machines can also be used to disassemble or remodel macromolecular complexes and to resolubilize aggregates.

Sculpting the Proteome with AAA+ Proteases and Disassembly Machines

Machines of protein destruction-including energy-dependent proteases and disassembly chaperones of the AAA(+) ATPase family-function in all kingdoms of life to sculpt the cellular proteome, ensuring that unnecessary and dangerous proteins are eliminated and biological responses to environmental change are rapidly and properly regulated. Exciting progress has been made in understanding how AAA(+) machines recognize specific proteins as targets and then carry out ATP-dependent dismantling of the tertiary and/or quaternary structure of these molecules during the processes of protein degradation and the disassembly of macromolecular complexes.

Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines.

Hexameric ring-shaped ATPases of the AAA + (for ATPases associated with various cellular activities) superfamily power cellular processes in which macromolecular structures and complexes are dismantled or denatured, but the mechanisms used by these machine-like enzymes are poorly understood. By covalently linking active and inactive subunits of the ATPase ClpX to form hexamers, here we show that diverse geometric arrangements can support the enzymatic unfolding of protein substrates and translocation of the denatured polypeptide into the ClpP peptidase for degradation. These studies indicate that the ClpX power stroke is generated by ATP hydrolysis in a single subunit, rule out concerted and strict sequential ATP hydrolysis models, and provide evidence for a probabilistic sequence of nucleotide hydrolysis. This mechanism would allow any ClpX subunit in contact with a translocating polypeptide to hydrolyse ATP to drive substrate spooling into ClpP, and would prevent stalling if one subunit failed to bind or hydrolyse ATP. Energy-dependent machines with highly diverse quaternary architectures and molecular functions could operate by similar asymmetric mechanisms.

Dynamics of Substrate Denaturation and Translocation by the ClpXP Degradation Machine

ClpXP is a protein machine composed of the ClpX ATPase, a member of the Clp/Hsp100 family of remodeling enzymes, and the ClpP peptidase. Here, ClpX and ClpXP are shown to catalyze denaturation of GFP modified with an ssrA degradation tag. ClpX translocates this denatured protein into the proteolytic chamber of ClpP and, when proteolysis is blocked, also catalyzes release of denatured GFP-ssrA from ClpP in a reaction that requires ATP and additional substrate. Kinetic experiments reveal that multiple reaction steps require collaboration between ClpX and ClpP and that denaturation is the rate-determining step in degradation. These insights into the mechanism of ClpXP explain how it executes efficient degradation in a manner that is highly specific for tagged proteins, irrespective of their intrinsic stabilities.

SmpB, a unique RNA-binding protein essential for the peptide-tagging activity of SsrA (tmRNA).

In bacteria, SsrA RNA recognizes ribosomes stalled on defective messages and acts as a tRNA and mRNA to mediate the addition of a short peptide tag to the C‐terminus of the partially synthesized nascent polypeptide chain. The SsrA‐tagged protein is then degraded by C‐terminal‐specific proteases. SmpB, a unique RNA‐binding protein that is conserved throughout the bacterial kingdom, is shown here to be an essential component of the SsrA quality‐control system. Deletion of the smpB gene in Escherichia coli results in the same phenotypes observed in ssrA‐defective cells, including a variety of phage development defects and the failure to tag proteins translated from defective mRNAs. Purified SmpB binds specifically and with high affinity to SsrA RNA and is required for stable association of SsrA with ribosomes in vivo. Formation of an SmpB–SsrA complex appears to be critical in mediating SsrA activity after aminoacylation with alanine but prior to the transpeptidation reaction that couples this alanine to the nascent chain. SsrA RNA is present at wild‐type levels in the smpB mutant arguing against a model of SsrA action that involves direct competition for transcription factors.

Linkage between ATP Consumption and Mechanical Unfolding during the Protein Processing Reactions of an AAA+ Degradation Machine

Proteolytic machines powered by ATP hydrolysis bind proteins with specific peptide tags, denature these substrates, and translocate them into a sequestered compartment for degradation. To determine how ATP is used during individual reaction steps, we assayed ClpXP degradation of ssrA-tagged titin variants with different stabilities in native and denatured forms. The rate of ATP turnover was 4-fold slower during denaturation than translocation. Importantly, this reduced turnover rate was constant during denaturation of native variants with different stabilities, but total ATP consumption increased with substrate stability, suggesting an iterative application of a uniform, mechanical unfolding force. Destabilization of substrate structure near the degradation tag accelerated degradation and dramatically reduced ATP consumption, revealing an important role for local protein stability in resisting denaturation. The ability to denature more stable proteins simply by using more ATP endows ClpX with a robust unfolding activity required for its biological roles in degradation and complex disassembly.

DNA recognition by beta-sheets in the Arc repressor-operator crystal structure

TRANSCRIPTION of the ant gene during lytic growth of bacteriophage P22 (ref. 1) is regulated by the cooperative binding of two Arc repressor dimers to a 21-base-pair operator site2,3. Here we report the co-crystal structure of this Arc tetramer–operator complex at 2.6 Å resolution. As expected from genetic4–6 and structural studies7 and from the co-crystal structure of the homologous Escherichia coli MetJ repressor8, each Arc dimer uses an antiparallel ß-sheet to recognize bases in the major groove. However, the Arc and Met J complexes differ in several important ways: the (β-sheet–DNA interactions of Arc are far less symmetrical; DNA binding by Arc is accompanied by important conformational changes in the β-sheet; and Arc uses a different part of its protein surface for dimer–dimer interactions.