Inosine-5'-monophosphate dehydrogenase (IMPDH), a key regulatory enzyme in purine nucleotide biosynthesis, dynamically assembles filaments in response to changes in metabolic demand. Humans have two ...isoforms: IMPDH2 filaments reduce sensitivity to feedback inhibition, while IMPDH1 assembly remains uncharacterized. IMPDH1 plays a unique role in retinal metabolism, and point mutants cause blindness. Here, in a series of cryogenic-electron microscopy structures we show that human IMPDH1 assembles polymorphic filaments with different assembly interfaces in extended and compressed states. Retina-specific splice variants introduce structural elements that reduce sensitivity to GTP inhibition, including stabilization of the extended filament form. Finally, we show that IMPDH1 disease mutations fall into two classes: one disrupts GTP regulation and the other has no effect on GTP regulation or filament assembly. These findings provide a foundation for understanding the role of IMPDH1 in retinal function and disease and demonstrate the diverse mechanisms by which metabolic enzyme filaments are allosterically regulated.
Small macrocycles with four or fewer amino acids are among the most potent natural products known, but there is currently no way to systematically generate such compounds. We describe a computational ...method for identifying ordered macrocycles composed of alpha, beta, gamma, and 17 other amino acid backbone chemistries, which we used to predict 14.9 million closed cycles composed of >42,000 monomer combinations. We chemically synthesized 18 macrocycles predicted to adopt single low-energy states and determined their x-ray or nuclear magnetic resonance structures; 15 of these were very close to the design models. We illustrate the therapeutic potential of these macrocycle designs by developing selective inhibitors of three protein targets of current interest. By opening up a vast space of readily synthesizable drug-like macrocycles, our results should considerably enhance structure-based drug design.
Despite remarkable advances in the assembly of highly structured coordination polymers and metal–organic frameworks, the rational design of such materials using more conformationally flexible organic ...ligands such as peptides remains challenging. In an effort to make the design of such materials fully programmable, we first developed a computational design method for generating metal-mediated 3D frameworks using rigid and symmetric peptide macrocycles with metal-coordinating sidechains. We solved the structures of six crystalline networks involving conformationally constrained 6 to 12 residue cyclic peptides with C2, C3, and S2 internal symmetry and three different types of metals (Zn2+, Co2+, or Cu2+) by single-crystal X-ray diffraction, which reveals how the peptide sequences, backbone symmetries, and metal coordination preferences drive the assembly of the resulting structures. In contrast to smaller ligands, these peptides associate through peptide–peptide interactions without full coordination of the metals, contrary to one of the assumptions underlying our computational design method. The cyclic peptides are the largest peptidic ligands reported to form crystalline coordination polymers with transition metals to date, and while more work is required to develop methods for fully programming their crystal structures, the combination of high chemical diversity with synthetic accessibility makes them attractive building blocks for engineering a broader set of new crystalline materials for use in applications such as sensing, asymmetric catalysis, and chiral separation.
Peptide and protein bioconjugation technologies have revolutionized our ability to site-specifically or chemoselectively install a variety of functional groups for applications in chemical biology ...and medicine, including the enhancement of bioavailability. Here, we introduce a site-specific bioconjugation strategy inspired by chemical ligation at serine that relies on a noncanonical amino acid containing a 1-amino-2-hydroxy functional group and a salicylaldehyde ester. More specifically, we harness this technology to generate analogues of glucagon-like peptide-1 that resemble Semaglutide, a long-lasting blockbuster drug currently used in the clinic to regulate glucose levels in the blood. We identify peptides that are more potent than unmodified peptide and equipotent to Semaglutide in a cell-based activation assay, improve the stability in human serum, and increase glucose disposal efficiency in vivo. This approach demonstrates the potential of “serine ligation” for various applications in chemical biology, with a particular focus on generating stabilized peptide therapeutics.
In response to the limited research experiences for young scholars during the COVID-19 pandemic and community interest, we developed the Pre-College Rosetta Internship Opportunity (PCR-IO). The ...mission of PCR-IO was to offer a program to increase equitable access to computational biomolecular research. The PCR-IO program engaged rising senior high school students in a protein therapeutic design project in which they produced novel structural models using the PyRosetta and Foldit software packages. The program comprised a year-long series of activities, with an immersive summer internship that involved students in research as the cornerstone. These activities aimed to support the overarching goal of the program by expanding participating students’ social capital and technical skills, making them more likely to consider and succeed in STEM in their future endeavors. Here we describe the program’s components and rollout and discuss successes and challenges in implementing a remote computational research-based educational high school program. We observed considerable student skill development and conclude that the program created real added value to student participants’ education. We also uncovered issues associated with curriculum pace and found that the required mentorship effort exceeded our expectations. This perspective is intended to offer insight, share recommendations, and create dialog to increase propagation of research-based computational internships, and to shed light on how much novice students can accomplish with mentorship, structured curricula, and access to the research community.
Cyclic peptides are a fascinating class of compounds to use in multiple design applications due to their small size and ability to be chemically synthesized. Non-canonical amino acids can be easily ...incorporated into cyclic peptides to expand their chemistries and make them resistant to proteolysis. Additionally, cyclic peptides are modular and can be computationally designed to adopt specific membrane permeable conformations. During my PhD, I used computationally designed cyclic peptides for two main applications, 1) designing peptide metal-organic frameworks, and 2) designing peptide inducible dimeric proteins.First, I explored the use of designed symmetric cyclic peptides to generate new metal-organic frameworks (MOFs). MOFs have shown great potential in a wide variety of applications such as small molecule separation, drug delivery, and catalysis. However, these materials have generally been limited to using small molecule linkers and short two to four residue peptides. I aimed to computationally design peptide MOFs using symmetric cyclic peptides. Peptide based MOFs provide advantages since they can bind specifically and selectively to different substrates and can be used for catalysis. We used the Rosetta Software Suit to dock and design peptides into metal mediated 3D lattices, then experimentally screened them for crystal formation. We solved the structures of six peptide materials using single crystal X-ray diffraction. Although these structures do not match the design models, they demonstrate fundamental thermodynamic and kinetic rules that govern the formation of such materials. Our computational pipeline is the first step to computationally design peptide based metal-organic frameworks and our experimental data provides information to further refine the computational protocol for more accurate modeling.Second, to gain insight into the effects of protein oligomerization on cellular functions, I employed cyclic peptides to induce the formation of protein oligomers. Most chemically inducible dimeric systems in the literature use clinically approved drugs to induce protein oligomerization. However, these drugs have off-target effects which makes the results ambiguous. In order to make CID systems that are orthogonal to cellular machinery, I used computationally designed de novo proteins and de novo cyclic peptides to make CIDs. I used Rosetta, Alphafold2, and ProteinMPNN to design and filter peptide binding proteins. The binding was validated using isothermal calorimetry, and equilibrium dialysis leading to the identification of multiple nanomolar and micromolar peptide binders. Finally, we can use the NanoBiT assay to optimize peptide-dependent protein oligomerization.
•VGF-derived peptides were synthesized and screened for effects on energy homeostasis.•NERP-1, PGH-NH2, HHPD-41, and TLQP-62 increased activity of hypothalamic networks.•ICV-delivered VGF-derived ...peptides did not alter energy homeostasis in vivo.•The data do not support VGF-derived peptides as anti-obesity drug candidates.
VGF is a peptide precursor expressed in neuroendocrine cells that is suggested to play a role in the regulation of energy homeostasis. VGF is proteolytically cleaved to yield multiple bioactive peptides. However, the specific actions of VGF-derived peptides on energy homeostasis remain unclear. The aim of the present work was to investigate the role of VGF-derived peptides in energy homeostasis and explore the pharmacological actions of VGF-derived peptides on body weight in preclinical animal models. VGF-derived peptides (NERP-1, NERP-2, PGH-NH2, PGH−OH, NERP-4, TLQP-21, TLQP-30, TLQP-62, HHPD-41, AQEE-30, and LQEQ-19) were synthesized and screened for their ability to affect neuronal activity in vitro on hypothalamic brain slices and modulate food intake and energy expenditure after acute central administration in vivo. In addition, the effects of NERP-1, NERP-2, PGH-NH2, TLQP-21, TLQP-62, and HHPD-41 on energy homeostasis were studied after chronic central infusion. NERP-1, PGH-NH2, HHPD-41, and TLQP-62 increased the functional activity of hypothalamic neuronal networks. However, none of the peptides altered energy homeostasis after either acute or chronic ICV administration. The present data do not support the potential use of the tested VGF-derived peptides as novel anti-obesity drug candidates.
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