Biology subjects

Rennella, E.

Publications and source records attributed to Rennella, E..

5 recordsLinked to original sources

bioRxiv2025

Assembly and substrate engagement mechanism of the bacterial proteasome activator Boa

The bacterial proteasomal activator Bpa (Rv3780) is an ATP-independent regulatory particle of the Mycobacterium tuberculosis proteasome system. Bpa recruits substrates as a dodecamer and triggers the gate opening of the proteasome 20S core particle; however, the structural basis for its oligomerization and substrate recognition remains unclear. Here, we define the temperature-sensitive oligomerization mechanism of Bpa and elucidate its interaction with a non-native substrate. Using size-exclusion chromatography, charge detection mass spectrometry, and pulsed hydrogen/deuterium exchange mass spectrometry (HDX-MS), we show that Bpa reversibly assembles into a dodecameric ring from dimeric and tetrameric species in a temperature-dependent manner. We used HDX-MS to map the oligomerization interfaces during Bpa assembly. Methyl transverse relaxation optimized spectroscopy (TROSY)-based NMR experiments and site-specific truncations further validate the existence of discrete tetrameric and dodecameric states. To overcome the limitations posed by the poor solubility of the native substrates of Bpa, we establish the DNA-binding domain of hTRF1 as a surrogate substrate. Bpa binds hTRF1 and mediates its degradation in a 20S CP-dependent manner. We quantify the affinity and stoichiometry of the Bpa-hTRF1 interaction using methyl-TROSY NMR, identifying a 12 Bpa subunit : 3 hTRF1 binding ratio with micromolar affinity that is modulated by salt concentration. Our NMR-based mapping experiments pinpoint the interaction surfaces on both Bpa and hTRF1, revealing key hydrophobic residues that mediate substrate engagement. This work uncovers a thermosensitive switch regulating Bpa oligomerization and activity and introduces a tractable substrate for dissecting proteasomal recognition in M. tuberculosis.

biochemistry↗

bioRxiv2024

Structural basis for allosteric regulation of the proteasome core particle

Intracellular protein degradation is vital across all domains of life1. In eukaryotes, the ubiquitin proteasome system performs most non-lysosomal protein degradation and influences numerous cellular processes. Some bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb), encode a proteasome system that selectively degrades damaged or misfolded proteins crucial for the pathogens survival within host macrophages2-7. Consequently, the 20S core particle (CP), the central component of the proteasome system, has emerged as a viable target for tuberculosis treatment strategies2,8-10. Both eukaryotic and Mtb proteasome systems are allosterically regulated11-13, yet the specific conformations involved have not been captured in high-resolution structures to date. Here we present the first structure of Mtb 20S CP, and indeed any 20S CP, in an inactive state called 20SOFF, distinguished from the canonical active state, 20SON, by the conformation of switch helices I and II. The rearrangement of these helices collapses the S1 pocket, effectively inhibiting substrate binding. The switch helices are conserved and regulate the activity of HslV protease, the proteasomes ancestral enzyme in bacteria, and a diverse family of serine/threonine protein phosphatases. Our results highlight the potential of harnessing allostery to develop therapeutics against the 20S CP in Mtb and eukaryotic systems.

biochemistry↗

bioRxiv2023

Design of amyloidogenic peptide traps

Segments of proteins with {beta}-strand propensity can self associate to form amyloid fibrils associated with many diseases. These regions often adopt alternative structures in their folded states, or are intrinsically disordered in solution, making it difficult to generate binders or inhibitors with existing strategies. Here we describe a general approach to bind such segments in {beta}-strand and {beta}-hairpin conformations using de novo designed scaffolds that contain deep peptide binding clefts flanked by {beta}-strands that form hydrogen bonds to the peptide upon binding. The designs bind their cognate peptides in vitro with nanomolar affinities and in mammalian cells. The crystal structure of a designed protein-peptide complex is close to the design model, and NMR characterization reveals how the peptide binding cleft is protected in the apo state. We use the approach to design binders to segments of the amyloid forming proteins Transthyretin, Tau, Serum amyloid A1 and A{beta}42. The A{beta} binders block assembly of A{beta} fibrils as effectively as the most potent of the clinically tested antibodies to date.

synthetic biology↗

bioRxiv2021

Opening of a Cryptic Pocket in β-lactamase Increases Penicillinase Activity

Understanding the functional role of protein excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in {beta}-lactamases and particularly a subset of states containing a cryptic pocket which forms under the {Omega}-loop. Given the known importance of the {Omega}-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol labeling assays to probe {Omega}-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across {beta}-lactamase homologs, those with the {Omega}-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM {beta}-lactamase variants with greater open {Omega}-loop pocket populations. We further investigate the open population using a combination of NMR CEST experiments and molecular dynamics simulations. To test our understanding of the {Omega}-loop pockets functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.

biophysics↗

bioRxiv2019

An allosteric switch regulates Mycobacterium tuberculosis ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR

The 300-kDa ClpP1P2 protease from Mycobacterium tuberculosis collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as novel antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of anti-TB therapeutics that target the Clp system. Here we present a combined biophysical and biochemical study to explore the structure-dynamics-function relationship in MtClpP1P2. Cryo-EM structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key {beta}-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that upon binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod-Wyman-Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function, and shows extensive allosteric interactions involving both intra- and inter-ring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target M. tuberculosis. Significance StatementThe MtClpP1P2 protease is part of the essential protein degradation machinery that helps maintain protein homeostasis in Mycobacterium tuberculosis, the causative agent of TB. Antibiotics that selectively kill both dormant and growing drug-resistant populations of M. tuberculosis by disrupting MtClpP1P2 function have attracted recent attention. Here we characterize a switch that can control MtClpP1P2 activity through binding of small peptides, leading to a concerted conformational change that potentially can be exploited by drug molecules to interfere with MtClpP1P2 function. Overall, this work highlights the power of a combined NMR and cryo-EM approach to provide detailed insights into the structure-dynamics-function relationship of molecular machines critical to human health.

biophysics↗