About

The Lisi laboratory uses solution NMR methods along with techniques in biochemistry, biophysics, and molecular biology to interrogate changes in protein structure and conformational motions that underlie function. With a focus on enzyme complexes, we aim to understand how biological events such as protein-protein interaction or the binding of allosteric effectors and drugs modulate protein motion, intra- and intermolecular signaling, and/or catalytic activity.

Research topics

• Biochemistry
• Computer Science
• Biology
• Computational chemistry
• Genetics
• Biophysics
• Computational biology
• Chemistry

Selected publications

• Exploring the GM-CSF Histidine Triad as a Modulator of Structure, Molecular Motion, and Ligand Binding

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-01-21

  articleOpen accessSenior authorCorresponding

  Granulocyte macrophage-colony stimulating factor (GM-CSF) is a cytokine that plays a role in immune modulation. Its expression is associated with a multitude of different effects ranging from harmful, as in diseases such as rheumatoid arthritis and multiple sclerosis, to beneficial, as in the case of mitigation of diabetes type I and neutropenia. However, there is a large gap in knowledge explaining how GM-CSF toggles its structure for such physiological and pathological interactions. Our work describes an ongoing attempt to address this gap by focusing on a clustered histidine triad within alpha-helices near the N-terminus, which prior studies have suggested play a role in binding ligands at an acidic pH. While GM-CSF is known to be highly flexible at a more acidic pH, several properties of its histidine triad remain unclear at the physiological pH at which GM-CSF would encounter its binding partners. We describe an effort to characterize the role of the GM-CSF histidines under physiological pH, specifically to determine if these histidines are key to GM-CSF structural integrity, and whether individual histidine residues modulate binding as they do at a lower pH. Our findings reveal that, while the histidine residues have an impact on GM-CSF structure, flexibility, and stability, they alone do not modulate the affinity for ligands at neutral pH. These data provide an initial explanation for the pleiotropic functions and interactions of GM-CSF within a biophysical context.

  PublisherOA PDFDOI

• Orthosteric and allosteric effects of anti-CRISPR II-C1 inhibition on <i>Geo</i> Cas9 from integrated structural biophysics

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-04-09

  articleOpen accessSenior authorCorresponding

  Abstract Anti-CRISPRs (Acrs) are small protein inhibitors of CRISPR-Cas effectors that originate from the translated genetic material of bacteriophage. Harnessing the natural ability of Acrs to bind and disrupt CRISPR-Cas editing can provide enhanced spatiotemporal control of gene editing. Recent studies have revealed diverse structures and functions of Acrs, however, atomistic studies of the specific molecular mechanisms behind Acr inhibition are lacking. Here, we reveal how structure, function, and dynamics govern AcrIIC1 inhibition of Cas9 from G. stearothermophilus ( Geo Cas9) via its HNH nuclease domain. An X-ray crystal structure of the Geo HNH-AcrIIC1complex reveals a conserved binding interface at the catalytic site and disruption of crucial electrostatic contacts known to modulate the thermostability of Geo Cas9. AcrIIC1 binding also rewires the intrinsic dynamics of the Geo HNH domain, stimulates millisecond motions that are absent from the unliganded nuclease, and attenuates the guide RNA affinity of Geo Cas9. Subsequent AcrIIC1 mutations in residues at its crystallographic binding interface uncouple Acr binding from inhibition, providing new insight into mechanism by which AcrIIC1 acts on Geo Cas9.

  PublisherOA PDFDOI

• Disorder, dynamics, and regulation of proteins and nucleic acids

  Journal of Structural Biology · 2026-01-05

  article1st authorCorresponding
  PublisherDOI

• Facilitating NMR Resonance Assignment with Metabolic Tampering

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-05-01

  articleOpen access

  The ability to assign amino acid resonances in multidimensional NMR spectra of biomolecules is necessary for detailed studies of protein structure and dynamics. Despite creative advances in isotopic labeling, unlabeling and multidimensional NMR experiments, resonance assignment remains a bottleneck in studies of large proteins. In this work, we show that the metabolic flux through biosynthetic pathways of amino acid production during protein expression can be modulated to aid in the identification of resonances in two-dimensional NMR spectra. This straightforward method involves doping 15N-enriched minimal media with small amounts of rich natural abundance media to generate unique peak intensity attenuation patterns, producing type-specific signatures of amino acids in two-dimensional 15N HSQC experiments. Using three model proteins, IGPS (51 kDa heterodimer), PTP1B (35 kDa), PHPT1 (14 kDa), we show that this method can disentangle several amino acid types, is robust to different expression conditions, and is a useful supplement for triple resonance experiments in protein backbone resonance assignments.

  PublisherDOI

• Discovery of Furan-2-Carboxylic Acid Derivatives as Novel D-Dopachrome Tautomerase (D-DT) and Macrophage Migration Inhibitory Factor-1 (MIF-1) Dual Inhibitors

  Journal of Medicinal Chemistry · 2026-03-02

  article

  D-dopachrome tautomerase (D-DT), also known as macrophage migration inhibitory factor-2, is a member of the MIF cytokine family and plays a key role in cancer and inflammation. Molecules that bind to the D-DT or MIF-1 tautomerase site could block their biological activity. However, relatively few D-DT inhibitors have been reported. In this study, we designed, synthesized, and screened a focused compound library. This led to the identification of 4h, a furan-2-carboxylic acid derivative with IC50 values of 2.4 μM for D-DT and 9.8 μM for MIF-1. Subsequent SAR optimization yielded the more potent inhibitor 10b, exhibiting IC50 values of 1.5 μM for D-DT and 1.0 μM for MIF-1. The specific interactions of 4h with D-DT and MIF-1 were explored using 1H–15N NMR endpoint titrations. 4h also inhibited D-DT-induced ERK phosphorylation in A549 cells. Thus, 4h and 10b represent a new class of inhibitors that can be utilized as tools to investigate the biological functions of D-DT and MIF-1.

  PublisherDOI

• BPS2026 – Biophysical principles for expanding PAM compatibility in CRISPR-Cas9 variants

  Biophysical Journal · 2026-02-01

  article
  PublisherDOI

• Comparative thermodynamic and kinetic properties governing the nucleic acid interactions of CRISPR-Cas9 and Cas12a

  Physical Biology · 2026-02-27

  articleOpen accessSenior author

  Clustered regularly interspaced short palindromic repeat-associated proteins (CRISPR-Cas) biochemistry has been leveraged for genome editing applications in biochemical research and therapeutics. CRISPR-Cas9 and CRISPR-Cas12a are the two most widely used RNA-guided endonucleases and while Cas9 and Cas12a have a shared function, both have unique biophysical properties that alter their specificity and efficiency. The thermodynamic and kinetic properties governing their molecular interactions, recognition and binding of target DNA, and R-loop formation can differ. In some cases, these critical biophysical metrics have not been resolved. Distinctions between Cas9 and Cas12a enzymes are also prevalent in RNA:DNA hybrid binding affinities, DNA localization relative to the preferred PAM site and the DNA cleavage mechanism. In this review, we examine the thermodynamic and kinetic properties of both endonucleases, focused on the nucleic acid interactions that confer specificity and function. Complementing this biophysical overview, we discuss case studies in disparate model organisms that compare the genome editing and fidelity of Cas9 and Cas12a.

  PublisherDOI

• Dynamic and structural insights into allosteric regulation on MKP5 a dual-specificity phosphatase

  Nature Communications · 2025-07-31 · 2 citations

  articleOpen accessSenior author

  Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (MKPs) directly dephosphorylate and inactivate the MAPKs. Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established, a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored. Here, we sought to define the molecular mechanism of MKP5 regulation through an allosteric site within its catalytic domain. We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y435 is required to maintain the structural integrity of the allosteric pocket. Along with molecular dynamics simulations, these data provide insight into how changes in the allosteric pocket propagate conformational flexibility in the surrounding loops to reorganize catalytically crucial residues in the active site. Furthermore, Y435 is required for the interaction with p38 MAPK and JNK, thereby promoting dephosphorylation. Collectively, these results demonstrate critical roles for the allosteric site in coordinating both MKP5 catalysis and MAPK binding. Authors show that the MKP5 phosphatase domain – required for catalysis – contains an allosteric site maintained by key hydrophobic residues, and this allosteric pocket binds MAPK which induces conformational changes that promote MAPK dephosphorylation.

  PublisherOA PDFDOI

• Design Rules for Expanding PAM Compatibility in CRISPR-Cas9 from the VQR, VRER and EQR variants

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-09-04

  preprintOpen access

  Expanding the range of Protospacer Adjacent Motifs (PAMs) recognized by CRISPR-Cas9 is essential for broadening genome-editing applications. Here, we combine molecular dynamics simulations with graph-theory and centrality analyses to dissect the principles of PAM recognition in three Cas9 variants - VQR, VRER, and EQR - that target non-canonical PAMs. We show that efficient recognition is not dictated solely by direct contacts between PAM-interacting residues and DNA, but also by a distal network that stabilizes the PAM-binding domain and preserves long-range communication with REC3, a hub that relays signals to the HNH nuclease. A key role emerges for the D1135V/E substitution, which enables stable DNA binding by K1107 and preserves key DNA phosphate locking interactions via S1109, securing stable PAM engagement. In contrast, variants carrying only R-to-Q substitutions at PAM-contacting residues, though predicted to enhance adenine recognition, destabilize the PAM-binding cleft, perturb REC3 dynamics, and disrupt allosteric coupling to HNH. Together, these findings establish that PAM recognition requires local stabilization, distal coupling, and entropic tuning, rather than a simple consequence of base-specific contacts. This framework provides guiding principles for engineering Cas9 variants with expanded PAM compatibility and improved editing efficiency.

  PublisherOA PDFDOI

• Design Rules for Expanding PAM Compatibility in CRISPR-Cas9 from the VQR, VRER and EQR variants

  The Journal of Physical Chemistry B · 2025-11-11 · 2 citations

  article

  Expanding the range of Protospacer Adjacent Motifs (PAMs) recognized by CRISPR-Cas9 is essential for broadening genome-editing applications. Here, we combine molecular dynamics simulations with graph-theory and centrality analyses to dissect the principles of PAM recognition in three Cas9 variants - VQR, VRER, and EQR - that target noncanonical PAMs. We show that efficient recognition is not dictated solely by direct contacts between PAM-interacting residues and DNA but also by a distal network that stabilizes the PAM-binding domain and preserves long-range communication with REC3, a hub that relays signals to the HNH nuclease. A key role emerges for the D1135 V/E substitution, which enables stable DNA binding by K1107 and preserves key DNA phosphate locking interactions via S1109, securing stable PAM engagement. In contrast, variants carrying only R-to-Q substitutions at PAM-contacting residues, though predicted to enhance adenine recognition, destabilize the PAM-binding cleft, perturb REC3 dynamics, and disrupt allosteric coupling to HNH. Together, these findings establish that PAM recognition requires local stabilization, distal coupling, and entropic tuning, rather than a simple consequence of base-specific contacts. This framework provides guiding principles for engineering Cas9 variants with expanded PAM compatibility and improved editing efficiency.

  PublisherDOI

Recent grants

• BioMedical Big Data Core

  NIH · $45.2M · 2016–2026

• Unraveling the Allosteric Mechanism of Macrophage Migration Inhibitory Factor with Molecular Resolution

  NIH · $1.3M · 2022–2027

• CAREER: Molecular Resolution of Long-range Allostery in CRISPR-Cas9

  NSF · $1.4M · 2022–2026

Frequent coauthors

• Erin Skeens

  Providence College

  85 shared

• G. Jogl

  Target (United States)

  55 shared

• Helen B. Belato

  Providence College

  51 shared

• Alexandra M. D’Ordine

  Providence College

  46 shared

• Víctor S. Batista

  Yale University

  42 shared

• Giulia Palermo

  37 shared

• Jennifer Y. Cui

  Providence College

  30 shared

• Kyle W. East

  Brown University

  27 shared

Labs

• Lisi Lab at Brown UniversityPI

  The Lisi lab studies the biochemistry and structural biology of enzyme and nucleic acid complexes.