Biology subjects

Krauss, I. J.

Publications and source records attributed to Krauss, I. J..

3 recordsLinked to original sources

bioRxiv2025

In vitro selection of cyclized, glycosylated peptide antigens that tightly bind HIV high mannose patch antibodies

In vitro selection is typically limited to discovery of peptides, proteins and nucleic acids. Given the importance of carbohydrate-protein interactions in diverse areas of biology including cell adhesion/recognition, immunoregulation and host-pathogen interactions, directed-evolution-based methods for discovery of potent glycoligands are greatly needed. We have previously reported a method for in vitro selection of glycopeptides that combines mRNA display, alkynyl amino acid incorporation, and CuAAC "click" glycosylation. Herein, we describe extensions of this method that incorporate chemical cyclization, removal of N-terminal glycosylation sites and next-generation sequencing; as an approach to HIV immunogen design, we have then used this method to develop mimics of the High Mannose Patch (HMP), which is the region on HIV envelope protein gp120 most commonly targeted by HIV broadly neutralizing antibodies (bnAbs). We prepared libraries of 1012-14 glycopeptides about 50 amino acids in length, containing variable numbers of high mannose (Man9GlcNAc2) glycans and cyclization at varied sites. We performed selections to obtain binders of HIV bnAbs PGT128, PGT122, and gl-PGT121, a germline precursor of PGT122, and prepared numerous glycopeptide hits by chemical synthesis. Selected glycopeptides in some cases bound very tightly to their target HIV bnAb, e.g., with a KD as low as 0.5 nM for PGT128. These glycopeptides are of interest as immunogens and tools for HIV vaccine design.

immunology↗

bioRxiv2024

Crystal Structure of Caryolan-1-ol Synthase, a Sesquiterpene Synthase Catalyzing an Initial Anti-Markovnikov Cyclization Reaction

In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from Streptomyces griseus, with and without an inactive analog of the FPP substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP complex was solved to 2.65 [A] resolution and showed the ligand in a linear, elongated orientation, incapable of undergoing the initial cyclization event to form a bond between carbons C1 and C11. Intriguingly, the apo CS structure (2.2 [A]) also had electron density in the active site, in this case density that was well fit with a curled-up tetraethylene glycol molecule presumably recruited from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotamer shifts among active-site residues. Most notably, W56 was observed to undergo a 90{degrees} rotation between the 2FFPP complex and apo-enzyme structures, suggesting that it contributes to steric interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L variant lost the ability to cyclize the FPP substrate and produced only the linear terpene products farnesol and - and {beta}-farnesene.

biochemistry↗

bioRxiv2020

Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase

Most terpene synthase reactions follow Markovnikov rules for formation of high energy carbenium ion intermediates. However, there are notable exceptions. For example, pentalenene synthase (PS) undergoes an initial anti-Markovnikov cyclization reaction followed by a 1,2-hydride shift to form an intermediate humulyl cation with positive charge on the secondary carbon C9 of the farnesyl diphosphate substrate. The mechanism by which these enzymes stabilize and guide regioselectivity of secondary carbocations has not heretofore been elucidated. In an effort to better understand these reactions, we grew crystals of apo-PS, soaked them with the non-reactive substrate analog 12,13-difluorofarnesyl diphosphate, and solved the x-ray structure of the resulting complex at 2.2 [A] resolution. The most striking feature of the active site structure is that C9 is positioned 3.5 [A] above the center of the side chain benzene ring of residue F76, perfectly poised for stabilization of the charge through a cation-{pi} interaction. In addition, the main chain carbonyl of I177 and neighboring intramolecular C6,C7-double bond are positioned to stabilize the carbocation by interaction with the face opposite that of F76. Mutagenesis experiments also support a role for residue 76 in cation-{pi} interactions. Most interesting is the F76W mutant which gives a mixture of products that likely result from stabilizing a positive charge on the adjacent secondary carbon C10 in addition to C9 as in the wild-type enzyme. The crystal structure of the F76W mutant clearly shows carbons C9 and C10 centered above the fused benzene and pyrrole rings of the indole side chain, respectively, such that a carbocation at either position could be stabilized in this complex, and two anti-Markovnikov products, pentalenene and humulene, are formed. Finally, we show that there is a rough correlation (although not absolute) of an aromatic side chain (F or Y) at position 76 in related terpene synthases from Streptomyces that catalyze similar anti-Markovnikov addition reactions.

biochemistry↗