About

Before closing his lab, Robert T. Sauer studied intracellular proteolytic machines responsible for protein-quality control and homeostasis. His research focused on the relationship between protein structure, function, sequence, and folding, specifically targeting the molecular machines that degrade or remodel proteins through ATP-dependent reactions. His experimental tools included biochemistry, single-molecule biophysics, structural biology, protein design and engineering, and molecular genetics. Sauer has made significant contributions to understanding the mechanisms of proteases such as ClpXP and HslUV, elucidating how these complexes recognize, unfold, and degrade protein substrates. His work has advanced the knowledge of protein degradation pathways and the structural basis of proteolytic machine function.

Research topics

• Biology
• Chemistry
• Cell biology
• Biochemistry
• Biophysics

Selected publications

• An asymmetric nautilus-like HflK/C assembly controls FtsH proteolysis of membrane proteins

  The EMBO Journal · 2025-03-13 · 21 citations

  articleOpen access

  The AAA protease FtsH associates with HflK/C subunits to form a megadalton-size complex that spans the inner membrane and extends into the periplasm of E. coli. How this bacterial complex and homologous assemblies in eukaryotic organelles recruit, extract, and degrade membrane-embedded substrates is unclear. Following the overproduction of protein components, recent cryo-EM structures showed symmetric HflK/C cages surrounding FtsH in a manner proposed to inhibit the degradation of membrane-embedded substrates. Here, we present structures of native protein complexes, in which HflK/C instead forms an asymmetric nautilus-shaped assembly with an entryway for membrane-embedded substrates to reach and be engaged by FtsH. Consistent with this nautilus-like structure, proteomic assays suggest that HflK/C enhances FtsH degradation of certain membrane-embedded substrates. Membrane curvature in our FtsH•HflK/C complexes is opposite that of surrounding membrane regions, a property that correlates with lipid scramblase activity and possibly with FtsH's function in the degradation of membrane-embedded proteins.

  PublisherOA PDFDOI

• Structural insights into the <i>Pseudomonas aeruginosa</i><scp>ClpP1</scp>•<scp>ClpP2</scp> heterocomplex and its interactions with the <scp>AAA</scp>+ <scp>ClpX</scp> unfoldase

  Protein Science · 2025-09-22 · 2 citations

  articleOpen accessSenior authorCorresponding

  Abstract ClpXP and other AAA+ proteases play central roles in bacterial proteostasis by degrading misfolded and regulatory proteins. In Pseudomonas aeruginosa , ClpXP consists of the ClpX unfoldase and ClpP peptidase, which influence critical adaptive processes contributing to stress resistance. P. aeruginosa Pa ClpP1 and Pa ClpP2 paralogs assemble into homomeric ( Pa ClpP1•ClpP1) and heteromeric ( Pa ClpP1•ClpP2) complexes. Pa ClpP2 is only active in the Pa ClpP1•ClpP2 heterocomplex. Here, we present a cryo‐EM structure of Pa ClpX•ClpP1•ClpP2, revealing how Pa ClpX binds Pa ClpP1, which in turn interacts with Pa ClpP2. Comparison of the active heterocomplex with an inactive Pa ClpP2 crystal structure shows that Pa ClpP1 binding induces conformational changes in Pa ClpP2, stabilizing an active catalytic triad. Differences in Pa ClpP1 and Pa ClpP2 substrate‐binding residues and an unstructured ClpP2 N‐terminal segment that protrudes into the peptidase chamber likely contribute to distinct peptide‐cleavage specificities of Pa ClpX•ClpP1•ClpP2 and Pa ClpX•ClpP1•ClpP1. Given the role of Pa ClpP1•ClpP2 in biofilm formation and virulence, these structural insights may provide a foundation for developing selective inhibitors to combat P. aeruginosa infections.

  PublisherOA PDFDOI

• An open axial channel of the <scp>AAA ClpXP</scp> protease enhances degradation of specific classes of protein substrates

  Protein Science · 2025-12-23

  articleOpen access

  ClpXP and other AAA proteases maintain proteostasis and regulate cellular functions by degrading misfolded, incomplete, or regulatory proteins. ClpX recognizes substrates via unstructured degron sequences, typically located at the N- or C-terminus. Although five classes of degrons are known, only recognition of the ssrA tag, a C-motif-1 degron, is well understood. The ssrA tag initially binds to a conformation of hexameric ClpX in which the axial channel is closed by a pore-2 loop, with subsequent channel opening allowing translocation into and degradation by ClpP. A ClpX variant with a pore-2 loop deletion (ΔNPS) favors the open conformation and exhibits weaker binding to ssrA-tagged substrates. Here, using model substrates representing each of the five known degron classes, we show that ΔNPS ClpXP degrades low micromolar concentrations of N-motif-1, -2, -3, and C-motif-2 substrates more effectively than wild-type ClpXP. Cryo-EM analysis of wild-type ClpXP bound to an N-motif-1 substrate reveals degron engagement within an open axial channel. Our results support a model in which the open- and closed-channel conformations of ClpXP differentially enhance recognition of distinct degron classes: the open channel facilitates degradation of many natural substrates, whereas the closed channel promotes efficient recognition and degradation of ssrA-tagged proteins. We propose that the conformational equilibrium between these two states tunes ClpXP activity to balance broad substrate recognition with specificity, allowing cells to meet dynamic proteolytic demands and minimize off-target degradation.

  PublisherOA PDFDOI

• Regulation of the Essential Transmembrane AAA+ Protease FtsH by HflK/C Oligomeric Assembly

  Structural Dynamics · 2025-03-01 · 1 citations

  articleOpen access

  Membrane-anchored AAA+ proteases, such as FtsH, degrade membrane-bound and soluble substrates to maintain protein homeostasis and regulate cellular functions across a diverse array of organisms and organelles, including eubacteria, chloroplasts, mitochondria, and apicomplexan parasites. FtsH functions as a homohexamer, with each monomer comprising a AAA+ module, a zinc-peptidase, transmembrane helices, and a periplasmic domain. The AAA+ module is responsible for ATP-dependent unfolding of substrates and translocation of the unfolded polypeptide chain into the peptidase chamber for degradation. In E. coli, FtsH interacts with the membrane proteins HflK and HflC, which together form a 1.8-MDa protein assembly within the bacterial inner membrane. However, the intricacies of how this complex assembles and functions, including how it recruits and extracts membrane substrates from the lipid bilayer for degradation, remain unknown. We determined a series of cryo-EM structures of the native FtsH•HflK/C complex from E. coli that contain two FtsH hexamers associated with each asymmetric HflK/C nautilus shell-like assembly. Our structures reveal an opening in the HflKC assembly that we hypothesize aids in recruitment of membrane-protein substrates. To probe for the effect of HflK/C on the degradation of putative substrates, we applied pulse-labeling mass spectrometry to both wild-type and ΔhflK/C strains of E. coli and identified a series of proteins whose rates of degradation depend on the presence of HflK/C. Integrating single-particle cryo-EM, cryo-ET, liposome reconstitution, and proteomic data, we propose a novel model for the FtsH•HflK/C microdomain that posits that the opening within the assembly provides an entryway to the FtsH axial channel. We further suggest that membrane curvature observed in our detergent-free structures may give rise to membrane thinning, facilitating the efficient extraction of FtsH substrates from the lipid bilayer.

  PublisherDOI

• Global Migration: Alternative Views and Social Comparisons

  WORLD SCIENTIFIC eBooks · 2024-02-28

  book-chapter1st authorCorresponding
  PublisherDOI

• A proteolytic AAA+ machine poised to unfold protein substrates

  Nature Communications · 2024-11-08 · 18 citations

  articleOpen access

  AAA+ proteolytic machines unfold proteins before degrading them. Here, we present cryoEM structures of ClpXP-substrate complexes that reveal a postulated but heretofore unseen intermediate in substrate unfolding/degradation. A ClpX hexamer draws natively folded substrates tightly against its axial channel via interactions with a fused C-terminal degron tail and ClpX-RKH loops that flexibly conform to the globular substrate. The specific ClpX-substrate contacts observed vary depending on the substrate degron and affinity tags, helping to explain ClpXP’s ability to unfold/degrade a wide array of different cellular substrates. Some ClpX contacts with native substrates are enabled by upward movement of the seam subunit in the AAA+ spiral, a motion coupled to a rearrangement of contacts between the ClpX unfoldase and ClpP peptidase. Our structures additionally highlight ClpX’s ability to translocate a diverse array of substrate topologies, including the co-translocation of two polypeptide chains. AAA proteases must unfold substrates before degradation. Here the authors report cryo-EM structures to visualize how these machines achieve this by pulling the substrate tightly against their narrow axial channel and encircling the substrate’s folded domain using only the unfoldase’s flexible loops.

  PublisherOA PDFDOI

• The membrane-cytoplasmic linker defines activity of FtsH proteases in Pseudomonas aeruginosa clone C

  Journal of Biological Chemistry · 2024-01-03 · 6 citations

  articleOpen access

  Pandemic Pseudomonas aeruginosa clone C strains encode two inner-membrane associated ATP-dependent FtsH proteases. PaftsH1 is located on the core genome and supports cell growth and intrinsic antibiotic resistance, whereas PaftsH2, a xenolog acquired through horizontal gene transfer from a distantly related species, is unable to functionally replace PaftsH1. We show that purified PaFtsH2 degrades fewer substrates than PaFtsH1. Replacing the 31-amino acid-extended linker region of PaFtsH2 spanning from the C-terminal end of the transmembrane helix-2 to the first seven highly divergent residues of the cytosolic AAA+ ATPase module with the corresponding region of PaFtsH1 improves hybrid-enzyme substrate processing in vitro and enables PaFtsH2 to substitute for PaFtsH1 in vivo. Electron microscopy indicates that the identity of this linker sequence influences FtsH flexibility. We find membrane-cytoplasmic (MC) linker regions of PaFtsH1 characteristically glycine-rich compared to those from FtsH2. Consequently, introducing three glycines into the membrane-proximal end of PaFtsH2's MC linker is sufficient to elevate its activity in vitro and in vivo. Our findings establish that the efficiency of substrate processing by the two PaFtsH isoforms depends on MC linker identity and suggest that greater linker flexibility and/or length allows FtsH to degrade a wider spectrum of substrates. As PaFtsH2 homologs occur across bacterial phyla, we hypothesize that FtsH2 is a latent enzyme but may recognize specific substrates or is activated in specific contexts or biological niches. The identity of such linkers might thus play a more determinative role in the functionality of and physiological impact by FtsH proteases than previously thought.

  PublisherOA PDFDOI

• An asymmetric nautilus-like HflK/C assembly controls FtsH proteolysis of membrane proteins

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-08-10 · 8 citations

  preprintOpen accessCorresponding

  . How this complex and homologous assemblies in eukaryotic organelles recruit, extract, and degrade membrane-embedded substrates is unclear. Following overproduction of protein components, recent cryo-EM structures reveal symmetric HflK/C cages surrounding FtsH in a manner proposed to inhibit degradation of membrane-embedded substrates. Here, we present structures of native complexes in which HflK/C instead forms an asymmetric nautilus-like assembly with an entryway for membrane-embedded substrates to reach and be engaged by FtsH. Consistent with this nautilus-like structure, proteomic assays suggest that HflK/C enhances FtsH degradation of certain membrane-embedded substrates. The membrane curvature in our FtsH•HflK/C complexes is opposite that of surrounding membrane regions, a property that correlates with lipid-scramblase activity and possibly with FtsH's function in the degradation of membrane-embedded proteins.

  PublisherOA PDFDOI

• DDM extracted overexpressed HflK/C nautilus-like assembly

  EMPIAR dataset · 2024-10-20

  datasetOpen access

  EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

  PublisherDOI

• How the double-ring ClpAP protease motor grips the substrate to unfold and degrade stable proteins

  Journal of Biological Chemistry · 2024-10-05 · 1 citations

  articleOpen access

  Loops in the axial channels of ClpAP and other AAA+ proteases bind a short peptide degron connected by a linker to the N- or C-terminal residue of a native protein to initiate degradation. ATP hydrolysis then powers pore-loop movements that translocate these segments through the channel until a native domain is pulled against the narrow channel entrance, creating an unfolding force. Substrate unfolding is thought to depend on strong contacts between pore loops and a subset of amino acids in the unstructured sequence directly preceding the folded domain. Here, we identify such contact sequences that promote grip for ClpAP and use ClpA structures to place these sequences within ClpA's two AAA+ rings. The positions and chemical nature of certain residues within an unstructured segment that are positioned to interact with the D2 ring have major positive effects on substrate unfolding, whereas segments located within the D1 ring have little consequence. Within the D2-bound segment, two short elements are critical for accelerating degradation; one is at the "top" of D2 and consists of at least two properly positioned nonslippery residues. In contrast, the second D2 element, which can be as short as one residue, is positioned to contact pore loops near the "bottom" of this ring. Comparison with similar studies for ClpXP reveals that positioning a well-gripped substrate sequence within the major unfoldase motor is more important than its proximity to the folded domain and that charged, polar, and hydrophobic residues all contribute favorable contacts to substrate grip.

  PublisherOA PDFDOI

Recent grants

• Pre-Doctoral Training in Biological Sciences

  NIH · $64.8M · 1975–2021

• AAA+ proteolytic machines

  NIH · $17.5M · 1980–2024

• NIH Grant R37AI015706

  NIH · $4.7M · 2012

• Sequence Determinants of Protein Structure and Stability

  NIH · $3.5M · 1979–2022

• NIH Grant R01AI015706

  NIH · $4.5M · 2002

Frequent coauthors

• Tania A. Baker

  Massachusetts Institute of Technology

  268 shared

• Hugh D. Niall

  University of Nottingham

  36 shared

• Igor Levchenko

  Pirogov Russian National Research Medical University

  32 shared

• Jason K. Sello

  University of California, San Francisco

  28 shared

• Robert A. Grant

  Massachusetts Institute of Technology

  28 shared

• Karl R. Schmitz

  University of Delaware

  23 shared

• Carl O. Pabo

  Harvard University Press

  22 shared

• Julia M. Flynn

  University of Massachusetts Chan Medical School

  20 shared

Labs

• Robert T. Sauer LabPI

Education

• Ph.D., Biochemistry and Molecular Biology

  Harvard University

  1979

• BA, Biophysics

  Amherst College

  1972

Awards & honors

• Protein Society, Stein and Moore Award, 2013
• Protein Society, Hans Neurath Award, 2007
• Protein Society, Amgen Award, 2001
• National Academy of Sciences, Member, 1996
• American Academy of Arts and Sciences, Fellow, 1993