The Lisi laboratory utilizes 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. Broadly, there are two major research arms of the lab: 1) studies of cytokine structural ensembles that dictate functional promiscuity and 2) molecular mapping of allosteric networks in large protein-nucleic acid complexes, such as CRISPR-Cas systems. Some highlights of each aspect are presented below.
- Integrated NMR and computational methodologies to resolve dynamic ensembles
The propagation of chemical signals via intrinsic protein dynamics plays a critical role in biology, though questions remain about the evolutionary nature of dynamic regulation and how the exact composition and interconversion rates of structural ensembles control ligand binding, allostery, and catalysis. The ability to accurately capture biomolecular motion is therefore essential to understanding modern protein function and its disruption in disease. Solution NMR can detect intrinsic structural flexibility on picosecond-to-second timescales that facilitates intra- and interprotein crosstalk. When coupled to molecular dynamics (MD) and network theory in silico, these methods can accurately define molecular motion and map the atomic level network network of communication. Our work leading and collaborating on these efforts have provided some enduring case studies for the application of experimental and theoretical concepts. More recent efforts have focused on conformational ensemble predictions via emerging AI like AlphaFold that can be validated with NMR.
- Monteiro da Silva, G.; Cui, J.Y.; Dalgarno, D.C.; Lisi, G.P.; Rubenstein, B.M.* High-throughput Prediction of Protein Conformational Distributions with Subsampled AlphaFold2 Nature Communications. 2024. 15. 2464-2476
- Skeens, E.; Lisi, G.P.* Analysis of Coordinated NMR Chemical Shifts to Map Allosteric Networks in Proteins Methods. 2023. 209. 40-47
- East, K.W.; Skeens, E.; Cui, J.Y.; Belato, H.B.; Mitchell, B.; Hsu, R.; Batista, V.S.; Palermo, G.; Lisi, G.P.* NMR and Computational Methods for Molecular Resolution of Allosteric Pathways in Enzyme Complexes Biophysical Reviews. 2020. 12. 155-174
- Skeens, E.; Lisi, G.P.* Analysis of Coordinated NMR Chemical Shifts to Map Allosteric Networks in Proteins Methods. 2023. 209. 40-47
- East, K.W.; Skeens, E.; Cui, J.Y.; Belato, H.B.; Mitchell, B.; Hsu, R.; Batista, V.S.; Palermo, G.; Lisi, G.P.* NMR and Computational Methods for Molecular Resolution of Allosteric Pathways in Enzyme Complexes Biophysical Reviews. 2020. 12. 155-174
- Environmental regulation of cytokine structure, ligand binding, and activity
Cytokines coordinate immunological and pro-inflammatory events through promiscuous cellular interactions and are associated with cancers, asthma, respiratory distress, and the "cytokine storm" of severe inflammation. Many of these pathological mechanisms are poorly understood, as are the biophysical properties that confer the ability of cytokines to engage in multiple non-overlapping functions. X-ray crystallographic studies of several cytokine families including structures containing mutations or ligands, appear identical despite changes in function. Paradoxically, NMR spectra of the same samples appear vastly different, suggesting NMR captures the true dynamic conformational equilibrium of these molecules in solution. We aim to understand how cytokines use conformational plasticity to spatially and temporally control biological signaling, focusing on the macrophage migration inhibitory factor (MIF) superfamily. Our work has mapped a network of amino acids that connect a known regulatory site in MIF to both a novel allosteric region and its biological signaling motif, which activates the CD74 receptor to promote inflammation. We have also confirmed this allosteric network to be a conserved property of the MIF superfamily, despite very little sequence homology. Our most recent work is exploring how oxidative solution conditions, a hallmark of inflammatory disease, modulates the structure, dynamics, and signaling mechanisms of MIF, as well as how redox-sensitive regions of MIF structure can be targeted with drugs.
- Sajko, S.; Skeens, E.; Schinagl, A.; Ferhat, M.; Mirkina, I.; Mayer, J.; Rossmueller, G.; Thiele, M.*; Lisi, G.P.* Redox-dependent Plasticity of oxMIF Facilitates its Interaction with CD74 and Therapeutic Antibodies Redox Biology. 2024. 75. 103264-103278
- Chen, E.; Reiss, K.; Shah, D.; Manjula, R.; Allen, B.; Murphy, E.L.; Murphy, J.W.; Batista, V.S.; Bhandari, V.; Lolis, E.J.*; Lisi, G.P.* A Structurally Preserved Allosteric Site in the MIF Superfamily Affects Enzymatic Activity and CD74 Activation in D-dopachrome Tautomerase J. Biol. Chem. 2021. 297. 101061-101073
- Pantouris, G.*; Khurana, L.; Ma, A.; Skeens, E.; Reiss, K.; Batista, V.S.; Lisi, G.P.*; Lolis, E.J.* Regulation of MIF Enzymatic Activity by an Allosteric Site at the Central Solvent Channel Cell Chem. Biol. 2020. 27. 740-750
- Chen, E.; Reiss, K.; Shah, D.; Manjula, R.; Allen, B.; Murphy, E.L.; Murphy, J.W.; Batista, V.S.; Bhandari, V.; Lolis, E.J.*; Lisi, G.P.* A Structurally Preserved Allosteric Site in the MIF Superfamily Affects Enzymatic Activity and CD74 Activation in D-dopachrome Tautomerase J. Biol. Chem. 2021. 297. 101061-101073
- Pantouris, G.*; Khurana, L.; Ma, A.; Skeens, E.; Reiss, K.; Batista, V.S.; Lisi, G.P.*; Lolis, E.J.* Regulation of MIF Enzymatic Activity by an Allosteric Site at the Central Solvent Channel Cell Chem. Biol. 2020. 27. 740-750
- Protein-drug interactions
Sites of flexibility in proteins can modulate biological signaling, catalytic mechanisms, and interactions with binding partners. Targeting small molecules to these flexible regions is an avenue to develop novel points of functional control in MIF and GM-CSF, and to establish signatures of "free" (black) and "bound" (red) states.
- Wu, Z.; Widjaja, V.; Skeens, E.; van der Velde, J.J.H.; Zahran, M.; Zhang, J.; Cool, R.H.; Poelarends, G.J.; Lisi, G.P.; Dekker, F.J.* 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. In press
- Widjaja, V.; D'Orazio, S.M.; Das, P.; Rajendran, D.T.; Takada, X.; Shi, Y.; Varghese, I.; Lam, Y.; DaSilva, N.; Wang, J.; Batista, V.S.; Bhandari, V.; Lisi, G.P.* Atomistic Modulation of MIF-2 Structure, Catalysis, and Biological Signaling via Cysteine Residues and a Small Molecule, Ebselen Protein Science. 2025. 34. e70344-e70359
- Cui, J.Y.; Zhang, F.; Nierzwicki, L.; Palermo, G.; Linhardt, R.J.; Lisi, G.P.* Mapping the Structural and Dynamic Determinants of pH-sensitive Heparin Binding to Granulocyte Macrophage-colony Stimulating Factor Biochemistry. 2020. 59. 3541-3553
- Widjaja, V.; D'Orazio, S.M.; Das, P.; Rajendran, D.T.; Takada, X.; Shi, Y.; Varghese, I.; Lam, Y.; DaSilva, N.; Wang, J.; Batista, V.S.; Bhandari, V.; Lisi, G.P.* Atomistic Modulation of MIF-2 Structure, Catalysis, and Biological Signaling via Cysteine Residues and a Small Molecule, Ebselen Protein Science. 2025. 34. e70344-e70359
- Cui, J.Y.; Zhang, F.; Nierzwicki, L.; Palermo, G.; Linhardt, R.J.; Lisi, G.P.* Mapping the Structural and Dynamic Determinants of pH-sensitive Heparin Binding to Granulocyte Macrophage-colony Stimulating Factor Biochemistry. 2020. 59. 3541-3553
- "Dynamic Allostery" - Molecular resolution of allosteric networks in CRISPR-Cas enzymes
The inability to fully explain allosteric regulation with classical paradigms clouds mechanistic aspects of proteins that are (de)activated via long-range "pathways" of intrinsic dynamics. A comprehensive understanding of how conformational fluctuations transmit chemical information through a protein matrix to induce a response is a "holy grail" of structural biology, but atomistic detail about specific amino acids comprising allosteric pathways that can be targeted as functional handles is lacking for most large proteins. Such insight has potential to make protein complexes intuitively controllable, lead to new mechanistic paradigms in fundamental biochemistry, and inform drug discovery, bioengineering, and precision medicine. We have performed NMR studies of CRISPR-Cas gene editing enzymes for many years, first revealing micro-millisecond structural flexibility in the nuclease domain as part of an allosteric pathway regulating cleavage of double-stranded DNA. Combined with MD simulations, NMR spin relaxation has traced functional connectivity through several Cas9 systems, providing insight into rewiring the Cas9 regulatory mechanism and active site architecture at the molecular level. We continue to explore the atomic aspect of CRISPR-Cas mechanisms, including its nucleic acid interactions and more recently, its inhibition.
- Belato, H.B.; Knight, A.L.; D'Ordine, A.M.; Pindi, C.; Fan, Z.; Luo, J.; Palermo, G.; Jogl, G.; Lisi, G.P.* Structural and Dynamic Impacts of Single-atom Disruptions to Guide RNA Interactions within the Recognition Lobe of Geobacillus stearothermophilus Cas9 eLife. 2024. 13. RP99275- RP99296
- Nierzwicki, L.; East, K.W.; Binz, J.; Hsu, R.V.; Arantes, P.R.; Ahsan, M.; Skeens, E.; Pacesa, M.; Jinek, M.; Lisi, G.P.*; Palermo, G.* Principles of Target DNA Cleavage and the Role of Mg2+ in the Catalysis of CRISPR-Cas9 Nature Catalysis. 2022. 5. 912-922
- East, K.W.; Newton, J.C.; Morzan, U.N.; Narkhede, Y.; Acharya, A.; Skeens, E.; Jogl, G.; Batista, V.S.; Palermo, G.*; Lisi, G.P.* Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics J. Am. Chem. Soc. 2020. 142. 1348-1358
- Nierzwicki, L.; East, K.W.; Binz, J.; Hsu, R.V.; Arantes, P.R.; Ahsan, M.; Skeens, E.; Pacesa, M.; Jinek, M.; Lisi, G.P.*; Palermo, G.* Principles of Target DNA Cleavage and the Role of Mg2+ in the Catalysis of CRISPR-Cas9 Nature Catalysis. 2022. 5. 912-922
- East, K.W.; Newton, J.C.; Morzan, U.N.; Narkhede, Y.; Acharya, A.; Skeens, E.; Jogl, G.; Batista, V.S.; Palermo, G.*; Lisi, G.P.* Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics J. Am. Chem. Soc. 2020. 142. 1348-1358
- Mechanisms of specificity enhancement in CRISPR-Cas9
The structural basis of specificity in nucleoprotein complexes is poorly understood and difficult to address. The reduction of CRISPR-Cas off-target activity via large mutational screens and error-prone PCR, while effective, is not intuitive. Thus, aspects of rational design of improved CRISPR-Cas variants are understudied. Allosteric communication between multiple domains of Cas9 is critical to its specificity, and specificity-enhancing mutations of many Cas9s are disproportionately localized to the nucleic acid recognition domain (REC), >10 Å from the nuclease active site. Our studies of such variants have revealed that, despite disparate locations, these mutations produce consistent structural patterns detectable by NMR, which propagate the allosteric signal of Cas9 in a manner distinct from wild-type. Several of our reports noted critical dynamic perturbations at regions of Cas9 that interface with its RNA:DNA hybrid to transduce chemical signals from REC to the catalytic domains. Many variants remodel the allosteric crosstalk across multiple domains by increasing communication between the since-identified DNA mismatch recognition helix and the nuclease active site. Our studies of the structural and dynamic underpinnings of Cas9 specificity has provided insight for future engineering principles.
- Vieyra, F.H.; Pindi, C.; Lisi, G.P.; Morzan, U.N.*; Palermo, G.* Design Rules for Expanding PAM Compatibility in CRISPR-Cas9 from the VQR, VRER, and EQR Variants Journal of Physical Chemistry B. 2025. 129. 11949-11958
- Skeens, E.; Sinha, S.; Ahsan, M.; D'Ordine, A.M.; Jogl, G.; Palermo, G.*; Lisi, G.P.* High-fidelity, Hyper-accurate, and Evolved Mutants Rewire Atomic Level Communication in CRISPR-Cas9 Science Advances. 2024. 10. eadl1045-1056
- Nierzwicki, L.; East, K.W.; Morzan, U.N.; Arantes, P.R.; Batista, V.S.; Lisi, G.P.*; Palermo, G.* Enhanced Specificity Mutations Perturb Allosteric Signaling in CRISPR-Cas9 eLife. 2021. 10. e73601
- Skeens, E.; Sinha, S.; Ahsan, M.; D'Ordine, A.M.; Jogl, G.; Palermo, G.*; Lisi, G.P.* High-fidelity, Hyper-accurate, and Evolved Mutants Rewire Atomic Level Communication in CRISPR-Cas9 Science Advances. 2024. 10. eadl1045-1056
- Nierzwicki, L.; East, K.W.; Morzan, U.N.; Arantes, P.R.; Batista, V.S.; Lisi, G.P.*; Palermo, G.* Enhanced Specificity Mutations Perturb Allosteric Signaling in CRISPR-Cas9 eLife. 2021. 10. e73601
- "Latent Allostery" - Uncovering new or dormant functional sites in protein complexes
The ability to exert functional control over an enzyme from a region that is spatially distinct from its active site is a biological approach that is becoming popular in drug discovery. Allosteric phenomena are widely acknowledged in biochemistry, but it remains unclear exactly how or why allostery manifests itself in enzymes. The expansive and complex nature of protein structures suggest that an overwhelming percentage of allosteric regulatory sites remain undiscovered. We are interested in exploring unifying (structural and dynamic) principles of allostery that can facilitate the identification of these sites a priori and elucidate new biomedically relevant molecular mechanisms. We are currently using a family of bacterial enzymes, phosphoribosyl anthranilate isomerases (PRAIs), that catalyze the same chemical reaction despite significant structural, oligomeric, and mechanistic diversity, as a model to understand the biochemical and biophysical features most critical to allostery.
- Ahsan, M.; Saha, A.; Ramos, D.; Strohkendl, I.; Skeens, E.; Lisi, G.P.; Taylor, D.W.; Palermo, G.* A Cryptic Binding Pocket Regulates the Metal-dependent Activity of Cas9 bioRxiv. 2025. DOI: 10.1101/2025.08.25.672025
- Skeens, E.; Gadzuk-Shea, M.M.; Shah, D.; Bhandari, V.; Schweppe, D.K.; Berlow, R.B.*; Lisi, G.P.* Redox-dependent Structure and Dynamics of Macrophage Migration Inhibitory Factor Reveal Sites of Latent Allostery Structure. 2022. 30. 840-850
- Parkins, A.; Skeens, E.; McCallum, C.M.; Lisi, G.P.*; Pantouris, G.* The N-terminus of MIF Regulates the Dynamic Profile of Residues Involved in CD74 Activation Biophys. J. 2021. 120. 1-8
- Skeens, E.; Gadzuk-Shea, M.M.; Shah, D.; Bhandari, V.; Schweppe, D.K.; Berlow, R.B.*; Lisi, G.P.* Redox-dependent Structure and Dynamics of Macrophage Migration Inhibitory Factor Reveal Sites of Latent Allostery Structure. 2022. 30. 840-850
- Parkins, A.; Skeens, E.; McCallum, C.M.; Lisi, G.P.*; Pantouris, G.* The N-terminus of MIF Regulates the Dynamic Profile of Residues Involved in CD74 Activation Biophys. J. 2021. 120. 1-8
