Biology subjects

Laganowsky, A.

Publications and source records attributed to Laganowsky, A..

11 recordsLinked to original sources

bioRxiv2026

Site Specific Hydration Contributes to the Thermal Stability of Proteins and Protein-Complexes

Water is integral to protein structure and stability, yet ensemble measurements rarely identify the hydration interactions associated with stabilization. Here, variable-temperature electrospray ionization ion mobility mass spectrometry, conformation-resolved thermodynamic analysis, and molecular dynamics simulations are combined to examine solvent-dependent thermal stability of chymotrypsin inhibitor 2 (CI-2), a model two-state folder, and the barnase-barstar (Bn-B*) complex, which contains a highly polar binding interface. Replacing H2O with D2O raises the thermal transition temperature of both systems by approximately 3 {degrees}C, favors compact conformations, and reduces the relative thermodynamic accessibility of extended states. The conformationally resolved enthalpy-entropy compensation reveals D2O increases enthalpy for CI-2 but reduces enthalpy for Bn-B* as a result of opposing effects on the individual subunits of the complex. Molecular dynamics simulations reveal that lower solvent-accessible surface areas and fewer protein-water hydrogen bonds in D2O coexist with longer water residence times at specific structural sites. In CI-2, the strongest changes localize to the second {beta}-strand/C-terminal region; in Bn-B*, persistent hydration is concentrated at the charged protein-protein interface. Together, these results support a model in which D2O dependent stabilization is associated not only with the extent of hydration, but the site and lifetime of water interactions. More broadly, this work provides an experimental framework for resolving solvent-dependent conformational thermodynamics of protein unfolding and interactions, enabling direct comparison of free-energy landscapes across solution environments.

biochemistry↗

bioRxiv2026

Beyond Structure: Protein and Solution Dynamics Shape Ligand-BindingThermodynamics

Variable-temperature electrospray ionization native mass spectrometry (vT-ESI nMS) provides a means to determine how temperature-dependent changes in protein and solution dynamics influence individual ligand-binding reactions within multiligand protein complexes. Here, the GroEL single-ring mutant (SR1), which binds up to seven nucleotides, serves as a model system due to its sensitivity to solution conditions. Native MS resolves individual ligand-bound populations, while vT-ESI extends these measurements across temperature (3-43 {degrees}C), enabling determination of binding affinities and the associated Gibbs free energy, enthalpy, entropy, heat capacity changes ({Delta}Cp), and enthalpy-entropy compensation (EEC) for each binding reaction. Temperature-dependent changes in van,t Hoff curvature produce distinct {Delta}Cp profiles, indicating changes in the dynamics governing ligand binding that may reflect contributions from protein conformational and protonation microstates. Temperature-dependent shifts in average charge state, consistent with changes in solvent-accessible surface area, indicate corresponding changes in protein conformation. Binding is predominantly enthalpy-driven below ~23 {degrees}C, with increasing entropic contributions at higher temperatures, while the seventh ADP binding reaction exhibits a distinct EEC profile. Comparison of the seventh ADP binding reaction in H2O and D2O reveals pronounced differences in van,t Hoff curvature,{Delta}Cp, and EEC, demonstrating that changes in the hydration environment alter the thermodynamic response. Collectively, these measurements show that temperature-dependent changes in protein and solution dynamics alter the distributions and thermodynamics of individual ligand-bound states and demonstrate the utility of vT-ESI nMS for resolving these effects in complex multiligand systems.

biochemistry↗

bioRxiv2026

Mechanism of substrate recognition and catalysis in a mammalian phosphatidylserine synthase 2

Mammalian phosphatidylserine synthase-1 and -2 synthesize phosphatidylserine (PS) by replacing the headgroup of either phosphatidylcholine (PC, PTDSS1) or phosphatidylethanolamine (PE, PTDSS2) with a serine. We determined structures of PTDSS2 from Equus caballus in complex with either PE or serine substrates to resolutions of 2.8-3.2 [A]. The structures define substrate binding sites and reveal that the phosphate group of PE is coordinated by two Ca2+. In addition, we found that PTDSS2 has significant phospholipase D (PLD) activity in the absence of serine, which was not reported previously, and that Ca2+ is required for the PLD activity. These discoveries enrich our knowledge in the mechanism of mammalian PTDSS.

biochemistry↗

bioRxiv2025

Molecular basis for the activation of Pseudomonas aeruginosa MsbA by Zn2+

Proteins involved in the biogenesis of lipopolysaccharide (LPS), a lipid exclusive to Gram-negative bacteria, are promising candidates for drug discovery. Specifically, the ABC transporter MsbA plays a crucial role in translocating an LPS precursor from the cytoplasmic to periplasmic facing leaflet of the inner membrane, and small molecules that inhibit its function exhibit bactericidal activity. Here, we use native mass spectrometry (MS) to determine lipid binding affinities of MsbA from P. aeruginosa (PaMsbA), a Gram-negative bacteria associated with hospital-acquired infections, in different conformations. We show the ATPase activity of the transporter is stimulated by Zn2+ and successfully trapping the protein with vanadate requires Zn2+ not Mg2+, which is necessary to trap MsbA from E. coli. We also present cryogenic-electron microscopy structures of PaMsbA in occluded and open outward-facing conformations determined to a resolution of 2.98 and 2.72 angstroms, respectively. The structures reveal a triad of histidine residues and mutation of these residues abolishes Zn2+ stimulation of PaMsbA activity. Together our studies provide detailed insight into PaMsbA structure, lipid binding preferences, and uncover a mechanism through which Zn2+ promotes the dimerization of the transporter, resulting in enhanced ATPase activity.

biochemistry↗

bioRxiv2024

Imperfect symmetry facilitated the evolution of specificity and high-order stoichiometry in vertebrate hemoglobin

Many proteins form paralogous multimers - molecular complexes in which evolutionarily related proteins are arranged into specific quaternary structures. Little is known about the mechanisms by which they acquired their stoichiometry (the number of total subunits in the complex) and heterospecificity (the preference of subunits for their paralogs rather than other copies of the same protein). Here we use ancestral protein reconstruction and biochemical experiments to study historical increases in stoichiometry and specificity during the evolution of vertebrate hemoglobin (Hb), a 2{beta}2 heterotetramer that evolved from a homodimeric ancestor after a gene duplication. We show that the mechanisms for this evolutionary transition were simple. One hydrophobic substitution in subunit {beta} after the gene duplication was sufficient to cause the ancestral dimer to homotetramerize with high affinity across a new interface. During this same interval, a single-residue deletion in subunit at the older interface conferred specificity for the heterotetrameric form and the trans-orientation of subunits within it. These sudden transitions in stoichiometry and specificity were possible because the interfaces in Hb are isologous - involving the same surface patch on interacting subunits, rotated 180{degrees} relative to each other. This architecture amplifies the impacts of individual mutations on stoichiometry and specificity, especially in higher-order complexes, and allows single substitutions to differentially affect heteromeric vs homomeric interactions. Our findings suggest that elaborate and specific symmetrical molecular complexes may often evolve via simple genetic and physical mechanisms. Significance statementMany molecular complexes are made up of proteins related by gene duplication, but how these assemblies evolve is poorly understood. Using ancestral protein reconstruction and biochemical experiments, we dissected how vertebrate hemoglobin, which comprises two copies each of two related proteins, acquired this architecture from a homodimeric ancestor. Each aspect of this transition - from dimer to tetramer and homomer to heteromer - had a simple genetic basis: a single-site amino acid change in each protein drove these changes in size and specificity. These transitions were possible because hemoglobins architecture is symmetric, which amplified the effect of small biochemical changes on the assembly of the complex. Many protein complexes are symmetrical, suggesting that they too may have evolved via simple genetic mechanisms. Classification: Major classification - Biological Sciences Minor classifications - Biochemistry/Evolution

evolutionary biology↗

bioRxiv2024

Native mass spectrometry of membrane protein-lipid interactions in different detergent environments

Native mass spectrometry (MS) is revealing the role of specific lipids in modulating membrane protein structure and function. Membrane proteins solubilized in detergents are often introduced into the mass spectrometer; however, commonly used detergents for structural studies, such as dodecylmaltoside, tend to generate highly charged ions, leading to protein unfolding, thereby diminishing their utility for characterizing protein-lipid interactions. Thus, there is a critical need to develop approaches to investigate protein-lipid interactions in different detergents. Here, we demonstrate how charge-reducing molecules, such as spermine and trimethylamine-N-oxide, enable characterization of lipid binding to the bacterial water channel (AqpZ) and ammonia channel (AmtB) in complex with regulatory protein GlnK in different detergent environments. We find protein-lipid interactions are not only protein-dependent but can also be influenced by the detergent and type of charge-reducing molecule. AqpZ-lipid interactions are enhanced in LDAO (n-dodecyl-N,N-dimethylamine-N-oxide), whereas the interaction of AmtB-GlnK with lipids is comparable among different detergents. A fluorescent lipid binding assay also shows detergent dependence for AqpZ-lipid interactions, consistent with results from native MS. Taken together, native MS will play a pivotal role in establishing optimal experimental parameters that will be invaluable for various applications, such as drug discovery, as well as biochemical and structural investigations.

biochemistry↗

bioRxiv2023

Double and triple thermodynamic mutant cycles reveal the basis for specific MsbA-lipid interactions

Structural and functional studies of the ATP-binding cassette transporter MsbA have revealed two distinct lipopolysaccharide (LPS) binding sites: one located in the central cavity and the other at a membrane-facing, exterior site. Although these binding sites are known to be important for MsbA function, the thermodynamic basis for these specific MsbA-LPS interactions is not well understood. Here, we use native mass spectrometry to determine the thermodynamics of MsbA interacting with the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL). The binding of KDL is solely driven by entropy, despite the transporter adopting an inward-facing conformation or trapped in an outward-facing conformation with adenosine 5-diphosphate and vanadate. An extension of the mutant cycle approach is employed to probe basic residues that interact with KDL. We find the molecular recognition of KDL is driven by a positive coupling entropy (as large as -100 kJ/mol at 298K) that outweighs unfavorable coupling enthalpy. These findings indicate that alterations in solvent reorganization and conformational entropy can contribute significantly to the free energy of protein-lipid association. The results presented herein showcase the advantage of native MS to obtain thermodynamic insight into protein-lipid interactions that would otherwise be intractable using traditional approaches, and this enabling technology will be instrumental in the life sciences and drug discovery.

biochemistry↗

bioRxiv2022

Binding of ATP to GroEL: a Case for Variable-Temperature Native Mass Spectrometry Thermodynamic Studies

Understanding how large, oligomeric protein complexes respond to the binding of small ligands and other proteins is essential for describing the molecular basis of life. This in turn requires a complete characterization of the binding energetics and correlation of thermodynamic data with interacting structures, including effects of small molecules and solvent. However, the size of many protein oligomers, the myriad intermediate ligation states they can populate, and their often complex allosteric regulation typically restrict analysis by traditional methods to low resolution, ensemble averages. Here, we employ variabletemperature electrospray ionization native mass spectrometry to determine the thermodynamics for stepwise binding of up to ATP molecules to the 801 kDa GroEL complex, a tetradecamer chaperonin complex. Binding thermodynamics reveal strong enthalpy-entropy compensation (EEC) and high degrees of cooperativity are observed for formation of GroEL-ATP7 and GroEL-ATP14. These are evidenced by entropically favored ATP binding to the cis ring (formation of GroEL-ATP1-7), with variations in EEC for subsequent binding of ATP to the trans ring (GroEL-ATP8-14), as expected for negative inter-ring cooperativity. Entropy driven ATP binding to the GroEL tetradecamer is consistent with ligand induced conformational changes of the GroEL tetradecamer, though the magnitude of the entropy change suggests that reorganization of GroEL-hydrating water molecules and/or expulsion of water from the GroEL cavity may also play a key role. By determining the thermodynamic signatures for individual ligand binding reactions to the large, nearly MDa GroEL complex, we expand our fundamental view of chaperonin functional chemistry. Moreover, this work and related studies of protein-ligand interactions illustrate unparalleled capabilities of vT-ESI-nMS for thermodynamics studies of protein interactions with ligands, and other molecules, such as proteins and drugs.

biophysics↗

bioRxiv2022

Structural basis for lipid and copper regulation of the ABC transporter MsbA

A critical step in Lipopolysaccharide (LPS) biogenesis involves flipping lipooligosaccharide, an LPS precursor, from the cytoplasmic to the periplasmic leaflet of the inner membrane, an operation carried out by the ATP-binding cassette transporter MsbA. Although MsbA has been extensively studied, the selectivity of MsbA-lipid interactions remains poorly understood. Here we use native mass spectrometry (MS) to characterize MsbA-lipid interactions and guide structural studies. We show the transporter co-purifies with copper(II) and metal binding modulates protein-lipid interactions. A 2.15 [A] resolution structure of an N-terminal region of MsbA in complex with copper(II) is presented, revealing a structure reminiscent of the GHK peptide, a high-affinity copper(II) chelator. Our results demonstrate conformation-dependent lipid binding affinities, particularly for the LPS-precursor, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KLA). We report a 3.6 [A]-resolution structure of MsbA trapped in an open, outward-facing conformation with adenosine 5-diphosphate and vanadate, revealing an unprecedented KLA binding site, wherein the lipid forms extensive interactions with the transporter. Additional studies provide evidence that the exterior KLA binding site is conserved and a positive allosteric modulator of ATPase activity, serving as a feedforward activation mechanism to couple transporter activity with LPS biosynthesis.

biochemistry↗

bioRxiv2020

A Sec14-like Phosphatidylinositol Transfer Protein Paralog Defines a Novel Class of Heme-binding Proteins With An Unusual Heme Coordination Mechanism

Yeast Sfh5 is an unusual member of the Sec14-like phosphatidylinositol transfer protein (PITP) family. Whereas PITPs are defined by their abilities to transfer phosphatidylinositol between membranes in vitro, and to stimulate phosphoinositide signaling in vivo, Sfh5 does not exhibit these activities. Rather, Sfh5 is a redox-active penta-coordinate high spin FeIII heme-binding protein with an unusual heme-binding arrangement that involves a co-axial tyrosine/histidine coordination strategy and a complex electronic structure connecting the open shell iron d-orbitals with three aromatic ring systems. That Sfh5 is not a PITP is supported by demonstrations that heme is not a readily exchangeable ligand, and that phosphatidylinositol-exchange activity is resuscitated in heme binding-deficient Sfh5 mutants. The collective data identify Sfh5 as the prototype of a new class of fungal hemoproteins, and emphasize the versatility of the Sec14-fold as scaffold for translating the binding of chemically distinct ligands to the control of diverse sets of cellular activities.

biochemistry↗

bioRxiv2020

Structure and mechanism of a primate ferroportin

Ferroportin is the only cellular iron exporter in human and essential for iron homoeostasis. Mutations in ferroportin are associated with hemochromatosis or ferroportin diseases characterized by a paradoxical combination of anemia and abnormal accumulation of iron in cells. Ferroportin is also the target of hepcidin, which is a hormone that downregulates ferroportin activity. However, due to a lack of three-dimensional structures, the mechanism of iron transport by ferroportin and its regulation by hepcidin remains unclear. Here we present the structure of a ferroportin from the primate Philippine tarsier (TsFpn) at 3.0 [A] resolution determined by cryo-electron microscopy. TsFpn has a structural fold common to major facilitator superfamily of transporters and the current structure is in an outward-open conformation. The structure identifies two potential ion binding sites with each site coordinated by two residues. Functional studies demonstrate that TsFpn is a H+/Fe2+ antiporter and that transport of one Fe2+ is coupled to the transport of two H+ in the opposite direction such that the transport cycle is electroneutral. Further studies show that the two ion binding sites affect transport of H+ and Fe2+ differently. The structure also provides mechanistic interpretation for mutations that cause ferroportin diseases.

biochemistry↗