Biology subjects

Rizo, A. N.

Publications and source records attributed to Rizo, A. N..

6 recordsLinked to original sources

bioRxiv2024

Design principles to tailor Hsp104 therapeutics

The hexameric AAA+ disaggregase, Hsp104, collaborates with Hsp70 and Hsp40 via its autoregulatory middle domain (MD) to solubilize aggregated protein conformers. However, how ATP- or ADP-specific MD configurations regulate Hsp104 hexamers remains poorly understood. Here, we define an ATP-specific network of interprotomer contacts between nucleotide-binding domain 1 (NBD1) and MD helix L1, which tunes Hsp70 collaboration. Manipulating this network can: (a) reduce Hsp70 collaboration without enhancing activity; (b) generate Hsp104 hypomorphs that collaborate selectively with class B Hsp40s; (c) produce Hsp70-independent potentiated variants; or (d) create species barriers between Hsp104 and Hsp70. Conversely, ADP-specific intraprotomer contacts between MD helix L2 and NBD1 restrict activity, and their perturbation frequently potentiates Hsp104. Importantly, adjusting the NBD1:MD helix L1 rheostat via rational design enables finely tuned collaboration with Hsp70 to safely potentiate Hsp104, minimize off- target toxicity, and counteract FUS proteinopathy in human cells. Thus, we establish important design principles to tailor Hsp104 therapeutics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/591398v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1520616org.highwire.dtl.DTLVardef@db3f5org.highwire.dtl.DTLVardef@1a1ae5eorg.highwire.dtl.DTLVardef@1beab7e_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Structures of the holo CRISPR RNA-guided transposon integration complex

CRISPR-associated transposons (CAST) are programmable mobile elements that insert large DNA cargo by an RNA-guided mechanism. Multiple conserved components act in concert at the target site through formation of an integration complex (transpososome). We reconstituted the type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined the structure using cryo-EM. Transpososome architecture ensures orientation-specific association: AAA+ regulator TnsC has defined polarity and length, with dedicated interaction interfaces for other CAST components. Interestingly, transposase (TnsB)-TnsC interactions we observe contribute to stimulating TnsCs ATP hydrolysis activity. TnsC deviates from previously observed helical configurations of TnsC, and target DNA does not track with TnsC protomers. Consequently, TnsC makes new, functionally important protein-DNA interactions throughout the transpososome. Finally, two distinct transpososome populations suggests that associations with the CRISPR effector are flexible. These ShCAST transpososome structures significantly enhances our understanding of CAST transposition systems and suggests avenues for improving CAST transposition for precision genome-editing applications.

biophysics↗

bioRxiv2022

Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps

The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-Spike-RBD and prevent viral entry into host cells ("receptor traps"). Here we determine cryo-EM structures of our receptor traps in complex with full length Spike. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high affinity (0.53 - 4.2nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron- and Delta-pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.

biochemistry↗

bioRxiv2021

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

biophysics↗

bioRxiv2019

Conformational Plasticity of the ClpAP AAA+ Protease Couples Protein Unfolding and Proteolysis

The ClpAP complex functions as a \"bacterial proteasome\" that simultaneously unfolds and degrades proteins targeted for destruction. ClpA utilizes two AAA+ domains per protomer to power substrate unfolding and translocation into the ClpP proteolytic chamber. To understand this mechanism, we determined high-resolution structures of wildtype E. coli ClpAP in distinct substrate-bound states. ClpA forms a spiral with substrate contacts across both AAA+ domains, while protomers at the seam undergo nucleotide-specific rearrangements indicating a conserved rotary mechanism. ClpA IGL loops extend flexibly to bind the planar, heptameric ClpP surface and support a large ClpA-P rotation that re-orients the translocation channel. The symmetry mismatch is maintained at the spiral seam through bind and release states of the IGL loops, which appear precisely coupled to substrate translocation. Thus, ClpA rotates around the apical surface of ClpP to processively translocate substrate into the protease.

biophysics↗

bioRxiv2018

Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase

Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. To elucidate the translocation mechanism, we determined the cryo-EM structure of a hyperactive ClpB variant to 2.9 [A] resolution bound to the model substrate, casein in the presence of slowly hydrolysable ATP{gamma}S. Distinct substrate-gripping mechanisms are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent the topmost NBD1 contact. NBD conformations at the spiral seam reveal how ATP hydrolysis and substrate engagement or disengagement are precisely tuned to drive a stepwise translocation cycle.

biophysics↗