Biology subjects

Silletti, S.

Publications and source records attributed to Silletti, S..

7 recordsLinked to original sources

bioRxiv2025

An allosteric network governs Tom70 conformational dynamics to coordinate mitochondrial import

Tom70 mediates mitochondrial protein import by coordinating the transfer of cytosolic preproteins from Hsp70/Hsp90 to the translocase of the outer membrane (TOM) complex. In humans, the cytosolic domain of Tom70 (HsTom70c) is entirely -helical and comprises modular TPR motifs divided into an N-terminal chaperone-binding domain (NTD) and a C-terminal preprotein-binding domain (CTD). However, the mechanisms linking these functional regions remain poorly understood. Here, we present the 2.04 [A] crystal structure of unliganded HsTom70c, revealing two distinct conformations - open and closed - within the asymmetric unit. These states are stabilized in part by interdomain crystal contacts and are supported in solution by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Principal component and dynamical network analyses reveal a continuum of motion linking the NTD and CTD via key structural elements, notably residues in helices 7, 8, and 25. Engagement of the CTD by the viral protein Orf9b interrupts this network, stabilizing a partially-closed intermediate conformation and dampening dynamics at distal NTD sites. Collectively, our findings lay the groundwork for understanding Tom70 allostery and provide a framework for dissecting its mechanistic roles in chaperone engagement, mitochondrial import, and viral subversion.

biophysics↗

bioRxiv2024

Active site remodeling in IDH1 mutants drives distinct kinetic and potential resistance mechanisms

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant uniquely preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling revealed a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

biochemistry↗

bioRxiv2022

Capturing differences in the regulation of LRRK2 dynamics and conformational states by small molecule kinase inhibitors

Mutations in the human leucine rich repeat protein kinase-2 (LRRK2) create risk factors for Parkinsons Disease, and pathological functions of LRRK2 are often correlated with aberrant kinase activity. Past research has focused on developing selective LRRK2 kinase inhibitors. We showed previously that in addition to influencing intrinsic kinase activity, the global conformation of the LRRK2 protein plays a vital role in regulating LRRK2 signaling pathways. Deciphering the allosteric regulation in LRRK2 provides novel strategies for drug discovery. In this study, we combined enhanced sampling simulations with HDX-MS to analyze the inhibitor-induced dynamic changes and the allosteric communications in the C-terminal half of LRRK2, LRRK2RCKW. We find that a type I inhibitor (MLi-2) locks the kinase into a closed, active-like configuration, whereas a type II inhibitor (Rebastinib) shifts the kinase to an open, inactive configuration. While both type I and type II inhibitors reduce the kinase activity effectively, they have distinct effects on the LRRK2 conformational dynamics. Specifically, binding of MLi-2 stabilizes the kinase domain in a closed conformation and reduces the global dynamics of LRRK2RCKW, leading to a more compact LRRK2RCKW structure. In contrast, binding of Rebastinib stabilizes an open conformation where communication between the N- and C-lobe is severed, which promotes a more extended LRRK2RCKW structure. Rebastinib, based on HDX-MS, creates a more dynamic kinase domain especially at domain interfaces associated with the C-lobe. Our results also reveal the importance of the Dk-helix, which plays a crucial role in propagating communication between the kinase domain and the GTPase domain.

biochemistry↗

bioRxiv2022

Target binding triggers hierarchical phosphorylation of human Argonaute-2 to promote target release

Argonaute (Ago) proteins play a central role in post-transcriptional gene regulation through RNA interference (RNAi). Agos bind small RNAs (sRNAs) including small interfering RNAs (siRNAs) and microRNAs (miRNAs) to form the functional core of the RNA Induced Silencing Complex (RISC). The sRNA is used as a guide to target mRNAs containing either partially or fully complementary sequences, ultimately leading to down regulation of the corresponding proteins. It was previously shown that the kinase CK1 phosphorylates a cluster of residues in the eukaryotic insertion (EI) of Ago, leading to the alleviation of miRNA-mediated repression through an undetermined mechanism. We show that binding of miRNA-loaded human Ago2 to target RNA with complementarity to the seed and 3 supplemental regions of the miRNA primes the EI for hierarchical phosphorylation by CK1. The added negative charges electrostatically promote target release, freeing Ago to seek out additional targets once it is dephosphorylated. The high conservation of potential phosphosites in the EI suggests that such a regulatory strategy may be a shared mechanism for regulating miRNA-mediated repression.

biochemistry↗

bioRxiv2020

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

In a multi-tiered approach, we explored how Parkinsons Disease-related mutations hijack the finely tuned activation process of Leucine-Rich Repeat Kinase 2 (LRRK2) using a construct containing the ROC, Cor, Kinase and WD40 domains (LRRK2RCKW). We hypothesized that the N-terminal domains shield the catalytic domains in an inactive state. PD mutations, type-I LRRK2 inhibitors, or physiological Rab GTPases can unleash the catalytic domains while the active kinase conformation, but not kinase activity, is essential for docking onto microtubules. Mapping solvent accessible regions of LRRK2RCKW employing hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed how inhibitor binding is sensed by the entire protein. Molecular Dynamics simulations of the kinase domain elucidated differences in conformational dynamics between wt and mutants of the DYG{psi} motif. While all domains contribute to regulating kinase activity and spatial distribution, the kinase domain, driven by the DYG{psi} motif, coordinates domain crosstalk and serves as an intrinsic hub for LRRK2 regulation.

biochemistry↗

bioRxiv2020

Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein

The COVID-2019 pandemic is the most severe acute public health threat of the twenty-first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS-CoV-2 coronavirus. Here, we examine the architecture and self-assembly properties of the SARS-CoV-2 nucleocapsid protein, which packages viral RNA into new virions. We determined a 1.4 [A] resolution crystal structure of this proteins N2b domain, revealing a compact, intertwined dimer similar to that of related coronaviruses including SARS-CoV. While the N2b domain forms a dimer in solution, addition of the C-terminal spacer B/N3 domain mediates formation of a homotetramer. Using hydrogen-deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an -helix that self-associates and cooperates with the N2b domain to mediate tetramer formation. Finally, we map the locations of amino acid substitutions in the N protein from over 38,000 SARS-CoV-2 genome sequences. We find that these substitutions are strongly clustered in the proteins N2a linker domain, and that substitutions within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self-assembly properties of a key protein in the SARS-CoV-2 life cycle, with implications for both drug design and antibody-based testing.

biochemistry↗

bioRxiv2019

Differential local stability governs the metamorphic fold-switch of bacterial virulence factor RfaH

A regulatory factor RfaH, present in many Gram-negative bacterial pathogens, is required for transcription and translation of long operons encoding virulence determinants. Escherichia coli RfaH action is controlled by a unique large-scale structural rearrangement triggered by recruitment to transcription elongation complexes through a specific DNA sequence within these operons. Upon recruitment, the C-terminal domain of this two-domain protein refolds from an -hairpin, which is bound to the RNA polymerase binding site within the N-terminal domain of RfaH, into an unbound {beta}-barrel that interacts with the ribosome to enable translation. Although structures of the autoinhibited (-hairpin) and active ({beta}-barrel) states and plausible refolding pathways have been reported, how this reversible switch is encoded within RfaH sequence and structure is poorly understood. Here, we combined hydrogen-deuterium exchange measurements by mass spectrometry and nuclear magnetic resonance with molecular dynamics to evaluate the differential local stability between both RfaH folds. Deuteron incorporation reveals that the tip of the C-terminal hairpin (residues 125-145) is stably folded in the autoinhibited state ([~]20% deuteron incorporation), while the rest of this domain is highly flexible (>40% deuteron incorporation) and its flexibility only decreases in the {beta}-folded state. Computationally-predicted {Delta}Gs agree with these results by displaying similar anisotropic stability within the tip of the -hairpin and on neighboring N-terminal domain residues. Remarkably, the {beta}-folded state shows comparable stability to non-metamorphic homologs. Our findings provide information critical for understanding the metamorphic behavior of RfaH and other chameleon proteins, and for devising targeted strategies to combat bacterial diseases.\n\nSignificanceInfections caused by Gram-negative bacteria are a worldwide health threat due to rapid acquisition of antibiotic resistance. RfaH, a protein essential for virulence in several Gram-negative pathogens, undergoes a large-scale structural rearrangement in which one RfaH domain completely refolds. Refolding transforms RfaH from an inactive state that restricts RfaH recruitment to a few target genes into an active state that binds to, and couples, transcription and translation machineries to elicit dramatic activation of gene expression. However, the molecular basis of this unique conformational change is poorly understood. Here, we combine molecular dynamics and structural biology to unveil the hotspots that differentially stabilize both states of RfaH. Our findings provide novel insights that will guide design of inhibitors blocking RfaH action.

biophysics↗