Biology subjects

Kollman, J. M.

Publications and source records attributed to Kollman, J. M..

At least 19 recordsLinked to original sources

bioRxiv2025

A Covalent PFKL Activator Suppresses Tumor Growth

Glycolysis fuels vital cellular functions and its dysregulation is implicated in cancer, neurodegeneration, antibiotic resistance and diabetes. The glycolytic dependency of cancer, known as the Warburg effect, presents a key vulnerability for developing targeted anticancer agents but remains challenging due to metabolic heterogeneity and resistance. Here, we developed a first-in-class covalent phosphofructokinase-1 liver type (PFKL) activator that induces metabolic imbalance coupled to delivery of a cytotoxic payload to cancer cells in vitro and in vivo. The electrophile-drug conjugate (EDC) site-specifically and proteome-wide selectively modifies K677 in the allosteric effector site to stabilize the R-state tetramer of PFKL and destabilize cell metabolism. We introduce EDCs as a new delivery mechanism analogous to antibody-drug conjugates but differentiated by selective covalent targeting of intracellular proteins.

biochemistry↗

bioRxiv2025

Product-stabilized filamentation by human glutamine synthetase allosterically tunes metabolic activity

To maintain metabolic homeostasis, enzymes must adapt to fluctuating nutrient levels through mechanisms beyond gene expression. Here, we demonstrate that human glutamine synthetase (GS) can reversibly polymerize into filaments aided by a composite binding site formed at the filament interface by the product, glutamine. Time-resolved cryo-electron microscopy (cryo-EM) confirms that glutamine binding stabilizes these filaments, which in turn exhibit reduced catalytic specificity for ammonia at physiological concentrations. This inhibition appears induced by a conformational change that remodulates the active site loop ensemble gating substrate entry. Metadynamics ensemble refinement revealed >10 [A] conformational range for the active site loop and that the loop is stabilized by transient contacts. This disorder is significant, as we show that the transient contacts which stabilize this loop in a closed conformation are essential for catalysis both in vitro and in cells. We propose that GS filament formation constitutes a negative-feedback mechanism, directly linking product concentration to the structural and functional remodeling of the enzyme.

bioinformatics↗

bioRxiv2025

An IMPDH2 variant associated with neurodevelopmental disorder disrupts purine biosynthesis and somitogenesis

IMP dehydrogenase (IMPDH) controls a key regulatory node in purine biosynthesis. Gain-of-function mutations in human IMPDH2 are associated with neurodevelopmental disorders and neuromuscular symptoms including dystonia, but the developmental mechanisms underlying these defects are unknown. We previously showed that these mutants are insensitive to GTP inhibition and hypothesized that their hyperactivity would affect nucleotide metabolism in vivo. Here, we characterize the metabolic and developmental consequences of the neurodevelopmental disorder-associated IMPDH2 mutant, S160del, in Xenopus tropicalis. We show that expressing S160del but not WT human IMPDH2 disrupts purine pools and somitogenesis in the developing tadpole. We also show that S160del disrupts in vivo IMPDH filament assembly, a well-described IMPDH regulatory mechanism. Cryo-EM structures show that S160del disrupts filament assembly by destabilizing the dimerization of regulatory Bateman domains. Dimerization of Bateman domains and subsequent filament formation can be restored with a high affinity ligand, but this does not restore sensitivity to GTP inhibition, suggesting S160del also disrupts allostery of IMPDH2 filaments. This work demonstrates that the structural effects of patient IMPDH2 variants can cause disruptions both to nucleotide levels and to the normal development of sensorimotor structures, helping us better understand the physiological basis of disease in these patients. SIGNIFICANCE STATEMENTIMPDH2 is a critical enzyme for de novo purine biosynthesis, regulating the balance between adenine and guanine nucleotides. Under purine stress, it forms filaments that resist feedback inhibition by GTP. Patients with gain-of-function variants of this enzyme suffer from early-onset neuromotor symptoms including dystonia. Here, we express one gain-of-function variant of IMPDH2, S160del, in Xenopus tropicalis. S160del is particularly powerful for structural and developmental studies, as it impedes filament formation and also is insensitive to feedback inhibition by GTP. Here, we show S160del can perturb vertebrate development, metabolism, and filament formation in a dominant fashion. Insights from this work will open the door to a new suite of studies defining the function of purine metabolism in development and disease.

biochemistry↗

bioRxiv2025

De novo design of inducibly assembling multi-component filaments

The design of multi-component nanomaterials is an outstanding challenge. Here, we describe the computational design of protein filaments with two or three distinct structural components that assemble into micron-scale, well-ordered fibers when mixed. CryoEM structure determination of four fiber designs was close to the computational design models. Filament assembly can be initiated by mixing the components, and modulated by addition and/or phosphorylation of designed regulatory subunits. This work demonstrates that regulatable multi-component protein filament systems can now be designed, opening the door to a wide range of engineered materials.

bioengineering↗

bioRxiv2024

Antibodies disrupt bacterial adhesion by ligand mimicry and allosteric interference

A critical step in infections is the attachment of many microorganisms to host cells using lectins that bind surface glycans, making lectins promising antimicrobial targets. Upon binding mannosylated glycans, FimH, the most studied lectin adhesin of type 1 fimbriae in E. coli, undergoes an allosteric transition from an inactive to an active conformation that can act as a catch-bond. Monoclonal antibodies that alter FimH glycan binding in various ways are available, but the mechanisms of these antibodies remain unclear. Here, we use cryoEM, mass spectrometry, binding assays, and molecular dynamics simulations to determine the structure-function relationships underlying antibody-FimH binding. Our study reveals four distinct antibody mechanisms of action: ligand mimicry by an N-linked, high-mannose glycan; stabilization of the ligand pocket in the inactive state; conformational trapping of the active and inactive states; and locking of the ligand pocket through long-range allosteric effects. These structures reveal multiple mechanisms of antibody responses to an allosteric protein and provide blueprints for new antimicrobial that target adhesins.

immunology↗

bioRxiv2024

Computational redesign of TALE proteins for DNA-templated assembly of protein fibers

Many viral proteins self-assemble into capsid structures, often using their genetic material as a template for assembly. To date, de novo designed capsid-like proteins do not require genetic material as a template for assembly, which can be both an advantage and a disadvantage depending on the use case. Templates are indispensable, for example, in the assembly of linear structures with well-defined lengths. As a first step towards fully de novo designed templated assembly, here we redesign proteins from the Transcription activator-like effector (TALE) family of transcriptional regulators to polymerize on double-stranded DNA (dsDNA) templates. Starting from natural TALE protein sequences, we created idealized repeat proteins with sequence-independent DNA binding properties that cooperatively self-assemble to form linear protein-DNA complexes with template-controlled lengths. We used high-resolution atomic force microscopy (AFM) and cryo electron microscopy (cryo-EM) to characterize the three-dimensional structures of the DNA-protein hybrid complexes. In these structures, a protein filament helically wraps around the dsDNA using a binding mode similar to that of natural TALE proteins. As an example application of these materials, we show the system can be used for repetitive peptide antigen display at precisely controlled repeat distances, and that such immunogens elicit robust antigen-specific antibodies in mice.

biochemistry↗

bioRxiv2024

Nucleation limited assembly and polarized growth of a de novo-designed allosterically modulatable protein filament

The design of inducibly assembling protein nanomaterials is an outstanding challenge. Here, we describe the computational design of a protein filament formed from a monomeric subunit that binds a peptide ligand. The cryoEM structure of the micron-scale fibers is very close to the computational design model. The ligand acts as a tunable allosteric modulator: while not part of the fiber subunit-subunit interfaces, the assembly of the filament is dependent on ligand addition, with longer peptides having more extensive interaction surfaces with the monomer, promoting more rapid growth. Seeded growth and capping experiments reveal that the filaments grow primarily from one end. We show that designed nucleators that present 12 copies of the peptide ligand promote fiber assembly at concentrations where otherwise assembly occurs very slowly, likely by generating critical local concentrations of monomer in the assembly competent conformation. Following filament assembly, the peptide ligand can be exchanged with free peptide in solution fused to any functional protein of interest, opening the door to a wide variety of tunable engineered materials.

bioengineering↗

bioRxiv2024

Appendage regeneration requires IMPDH2 and creates a sensitized environment for enzyme filament formation

Regeneration of lost tissue requires biosynthesis of metabolites needed for cell proliferation and growth. Among these are the critical purine nucleotides ATP and GTP. The abundance and balance of these purines is regulated by inosine monophosphate dehydrogenase 2 (IMPDH2), which catalyzes the committing step of GTP synthesis. IMPDH2 assembles into filaments that resist allosteric inhibition under conditions of high GTP demand. Here we asked whether IMPDH2 is required in the highly proliferative context of regeneration, and whether its assembly into filaments takes place in regenerating tissue. We find that inhibition of IMPDH2 leads to impaired tail regeneration and reduced cell proliferation in the tadpole Xenopus tropicalis. We find that both endogenous and fluorescent fusions of IMPDH2 robustly assemble into filaments throughout the tadpole tail, and that the regenerating tail creates a sensitized condition for filament formation. These findings clarify the role of purine biosynthesis in regeneration and reveal that IMPDH2 enzyme filament formation is a biologically relevant mechanism of regulation in vertebrate regeneration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/605679v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1006d64org.highwire.dtl.DTLVardef@10ddf85org.highwire.dtl.DTLVardef@1cdc99eorg.highwire.dtl.DTLVardef@16fc4cb_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

Evolutionarily divergent Mycobacterium tuberculosis CTP synthase filaments are under selective pressure

The final and rate-limiting enzyme in pyrimidine biosynthesis, CTP synthase (CTPS), is essential for the viability of Mycobacterium tuberculosis and other mycobacteria. Its product, CTP, is critical for RNA, DNA, lipid and cell wall synthesis, and is involved in chromosome segregation. In various organisms across the tree of life, CTPS assembles into higher-order filaments, leading us to hypothesize that M. tuberculosis CTPS (mtCTPS) also forms higher-order structures. Here, we show that mtCTPS does assemble into filaments but with an unusual architecture not seen in other organisms. Through a combination of structural, biochemical, and cellular techniques, we show that polymerization stabilizes the active conformation of the enzyme and resists product inhibition, potentially allowing for the highly localized production of CTP within the cell. Indeed, CTPS filaments localize near the CTP-dependent complex needed for chromosome segregation, and cells expressing mutant enzymes unable to polymerize are altered in their ability to robustly form this complex. Intriguingly, mutants that alter filament formation are under positive selection in clinical isolates of M. tuberculosis, pointing to a critical role needed to withstand pressures imposed by the host and/or antibiotics. Taken together, our data reveal an unexpected mechanism for the spatially organized production of a critical nucleotide in M. tuberculosis, which may represent a vulnerability of the pathogen that can be exploited with chemotherapy.

biochemistry↗

bioRxiv2024

Structural basis for allosteric regulation of human phosphofructokinase-1

Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of the cell. In bacteria, the structural basis of PFK1 regulation is a textbook example of allostery; molecular signals of low and high cellular energy promote transition between an active R-state and inactive T-state conformation, respectively. Little is known, however, about the structural basis for regulation of eukaryotic PFK1. Here, we determine structures of the human liver isoform of PFK1 (PFKL) in the R- and T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory mechanisms. The T-state structure reveals conformational differences between the bacterial and eukaryotic enzyme, the mechanisms of allosteric inhibition by ATP binding at multiple sites, and an autoinhibitory role of the C-terminus in stabilizing the T-state. We also determine structures of PFKL filaments that define the mechanism of higher-order assembly and demonstrate that these structures are necessary for higher-order assembly of PFKL in cells.

biochemistry↗

bioRxiv2023

A family of bacterial actin homologues forms a 3-stranded tubular structure

The cytoskeleton plays a critical role in the organization and movement of cells. In Eukaryotes, actin filaments polymerize into a highly conserved double-stranded linear filamentous structure in the presence of ATP, and disassemble upon ATP hydrolysis. Bacteria also possess actin-like proteins, that drive fundamental cellular function, including cell division, shape maintenance, and DNA segregation. Like eukaryotic actin, bacterial actins assemble upon ATP binding. Longitudinal interactions between bacterial actin protomers along each strand are conserved with eukaryotic actin, but variation in interactions between strands gives rise to striking diversity of filament architectures. Here, we report a family of bacterial actins of unknown function, conserved amongst the Verrucomicrobiota phylum, which assembles into a unique tubular structure in the presence of ATP. A cryo-EM structure of the filaments reveals that it consists of three strands, unlike other described bacterial actin structures. This architecture provides new insights into the organization of actin-like filaments, and has implications for understanding the diversity and evolution of the bacterial cytoskeleton.

biophysics↗

bioRxiv2023

Light-sensitive phosphorylation regulates enzyme activity and filament assembly of human IMPDH1 retinal splice variants

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanosine triphosphate (GTP) synthesis and is controlled by feedback inhibition and allosteric regulation. IMPDH assembles into micron-scale filaments in cells, which desensitizes the enzyme to feedback inhibition by GTP and boosts nucleotide production. The vertebrate retina expresses two tissue-specific splice variants IMPDH1(546) and IMPDH1(595). IMPDH1(546) filaments adopt high and low activity conformations, while IMPDH1(595) filaments maintain high activity. In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of phosphorylation in IMPDH1 variants. The S477D mutation re-sensitized both variants to GTP inhibition, but only blocked assembly of IMPDH1(595) filaments and not IMPDH1(546) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of the high activity assembly interface, still allowing assembly of low activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, phosphorylation at S477 acts as a mechanism for downregulating retinal GTP synthesis in the dark, when nucleotide turnover is decreased. Like IMPDH1, many other metabolic enzymes dynamically assemble filamentous polymers that allosterically regulate activity. Our work suggests that posttranslational modifications may be yet another layer of regulatory control to finely tune activity by modulating filament assembly in response to changing metabolic demands. SIGNIFICANCE STATEMENTOver 20 different metabolic enzymes form micron-scale filaments in cells, suggesting that filament assembly is a conserved mechanism for regulating diverse metabolic pathways. Filament assembly regulates catalytic activity of many of these enzymes, including inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo GTP biosynthesis. The vertebrate retina expresses two IMPDH1 splice variants that are critical for maintaining nucleotide levels required for phototransduction. Here, we show that filament assembly by these variants is itself controlled by phosphorylation at a single residue, adding further complexity to the tight regulation of nucleotide metabolism in the retina. Phosphorylation and other posttranslational modifications are likely to be a general regulatory mechanism controlling filament assembly by enzymes in many different metabolic pathways.

biochemistry↗

bioRxiv2023

Toxoplasma gondii actin filaments are tuned for rapid disassembly and turnover

The cytoskeletal protein actin plays a critical role in the pathogenicity of Toxoplasma gondii, mediating invasion and egress, cargo transport, and organelle inheritance. Advances in live cell imaging have revealed extensive filamentous actin networks in the Apicomplexan parasite, but there is conflicting data regarding the biochemical and biophysical properties of Toxoplasma actin. Here, we imaged the in vitro assembly of individual Toxoplasma actin filaments in real time, showing that native, unstabilized filaments grow tens of microns in length. Unlike skeletal muscle actin, Toxoplasma filaments intrinsically undergo rapid treadmilling due to a high critical concentration, fast monomer dissociation, and rapid nucleotide exchange. Cryo-EM structures of stabilized and unstabilized filaments show an architecture like skeletal actin, with differences in assembly contacts in the D-loop that explain the dynamic nature of the filament, likely a conserved feature of Apicomplexan actin. This work demonstrates that evolutionary changes at assembly interfaces can tune dynamic properties of actin filaments without disrupting their conserved structure.

biophysics↗

bioRxiv2023

Blueprinting expandable nanomaterials with standardized protein building blocks

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies in comparison has been much more complex, largely due to the irregular shapes of protein structures1. Here we describe extendable linear, curved, and angled protein building blocks, as well as inter-block interactions that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight "train track" assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not been previously possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank 3D canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to "back of an envelope" architectural blueprints.

biochemistry↗

bioRxiv2023

Point mutations in IMPDH2 which cause early-onset neurodevelopmental disorders disrupt enzyme regulation and filament structure

Inosine 5 monophosphate dehydrogenase (IMPDH) is a critical regulatory enzyme in purine nucleotide biosynthesis that is inhibited by the downstream product GTP. Multiple point mutations in the human isoform IMPDH2 have recently been associated with dystonia and other neurodevelopmental disorders, but the effect of the mutations on enzyme function has not been described. Here, we report identification of two additional affected individuals with missense variants in IMPDH2 and show that all of the disease-associated mutations disrupt GTP regulation. Cryo-EM structures of one IMPDH2 mutant suggest this regulatory defect arises from a shift in the conformational equilibrium toward a more active state. This structural and functional analysis provides insight into IMPDH2-associated disease mechanisms that point to potential therapeutic approaches and raises new questions about fundamental aspects of IMPDH regulation. Significance StatementPoint mutations in the human enzyme IMPDH2, a critical regulator of nucleotide biosynthesis, are linked to neurodevelopmental disorders, such as dystonia. Here, we report two additional IMPDH2 point mutants associated with similar disorders. We investigate the effects of each mutation on IMPDH2 structure and function in vitro and find that all mutations are gain of function, preventing allosteric regulation of IMPDH2 activity. We report high resolution structures of one variant and present a structure-based hypothesis for its dysregulation. This work provides a biochemical basis for understanding diseases caused by IMPDH2 mutation and lays a foundation for future therapeutic development.

biochemistry↗

bioRxiv2022

Human PRPS1 filaments stabilize allosteric sites to regulate activity

The universally conserved enzyme phosphoribosyl pyrophosphate synthetase (PRPS) assembles filaments in evolutionarily diverse organisms. PRPS is a key regulator of nucleotide metabolism, and mutations in the human enzyme PRPS1 lead to a spectrum of diseases. Here, we determine structures of PRPS1 filaments in active and inhibited states, with fixed assembly contacts accommodating both conformations. The conserved assembly interface stabilizes the binding site for the essential activator phosphate, increasing activity in the filament. Some disease mutations alter assembly, supporting the link between filament stability and activity. Structures of active PRPS1 filaments turning over substrate also reveal coupling of catalysis in one active site with product release in an adjacent site. PRPS1 filaments therefore provide an additional layer of allosteric control, conserved throughout evolution, with likely impact on metabolic homeostasis. Stabilization of allosteric binding sites by polymerization adds to the growing diversity of assembly-based enzyme regulatory mechanisms.

biochemistry↗

bioRxiv2021

Computational design of nanoscale rotational mechanics in de novo protein assemblies

Natural nanomachines like the F1/F0-ATPase contain protein components that undergo rotation relative to each other. Designing such mechanically constrained nanoscale protein architectures with internal degrees of freedom is an outstanding challenge for computational protein design. Here we explore the de novo construction of protein rotary machinery from designed axle and ring components. Using cryoelectron microscopy, we find that axle-ring systems assemble as designed and populate diverse rotational states depending on symmetry match or mismatch and the designed interface energy landscape. These mechanical systems with internal rotational degrees of freedom are a step towards the systematic design of genetically encodable nanomachines. One-Sentence SummaryComputationally designed self-assembling protein rotary machines sample internal degrees of freedom sculpted within the energy landscape.

synthetic biology↗

bioRxiv2021

Cryo-EM Structures of CTP Synthase Filaments Reveal Mechanism of pH-Sensitive Assembly During Budding Yeast Starvation

Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or hyper-assembly mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

biochemistry↗