Biology subjects

Kober, D. L.

Publications and source records attributed to Kober, D. L..

6 recordsLinked to original sources

bioRxiv2026

A domain-swapped proPC1/3 structure reveals a conformational checkpoint for ER export

Prohormone convertase 1/3 (PC1/3, encoded by PCSK1) is a serine protease expressed in neuroendocrine cells that is required to produce insulin, glucagon-like peptide-1, adrenocorticotrophic hormone, and other peptide hormones. PC1/3 is synthesized as the zymogen proPC1/3, which undergoes autocatalytic maturation in the endoplasmic reticulum (ER) before trafficking to secretory granules. However, how autocatalytic maturation licenses the ER exit of PC1/3 remains unknown. Here, we determined the structure of an immature, catalytically inactive human proPC1/3S382A, which exits the ER as a domain-swapped homodimer. This domain-swapped conformation allows proPC1/3S382A to complete a conserved calcium pocket normally formed after autocatalysis and thereby become competent for ER exit. By defining this calcium pocket as a conformational checkpoint for ER export, our findings provide insights into the maturation of PC1/3 which may apply broadly to the PCSK family. Finally, our structure provides explanations for the functional consequences of many deleterious PCSK1 mutations.

biochemistry↗

bioRxiv2025

Choreography of human mitochondrial leaderless mRNA translation initiation

It remains unclear how human mitochondrial ribosomal subunits assemble into an elongation-competent 55S particle on mRNAs devoid of 5 leader sequences. Here, we reconstituted and directly tracked human mitochondrial translation initiation using real-time single-molecule fluorescence spectroscopy. Corroborated with cryo-EM structural analysis, we show that the initiation factor mtIF2 and initiator fMet-tRNAMet are loaded to the 28S subunit to drive mRNAs binding via 5 start codon recognition. This enables sequential loading of the two ribosomal subunits onto the leaderless mRNA to initiate. In parallel, a preassembled 55S monosome can also be loaded with mtIF2 and fMet-tRNAMet to initiate on the mRNA. Both initiation pathways yield active complexes to enter translation elongation, which is gated by mtIF2. The monosome loading pathway can initiate promiscuously with non-formylated Met-tRNAMet, thus its usage may under tight regulation in cells, e.g. by mtIF3. Our work provides a dynamic framework for the distinct human mitochondrial translation initiation.

biochemistry↗

bioRxiv2025

A biochemical mechanism for Stu2/XMAP215-family microtubule polymerases

Defining quantitative biochemical mechanisms of microtubule dynamics and regulation is a current challenge. Stu2/XMAP215-family polymerases use tubulin-binding TOG domains to catalyze microtubule growth, but how polymerase activity results from the number and tubulinbinding properties of TOGs is not understood. We tested whether an enzyme-like biochemical model for the unrelated actin polymerase Ena/VASP could be applied to quantitatively relate Stu2 microtubule polymerase activity to the number of its TOGs and the rate constants governing their interactions with tubulin. Stu2 activity displayed enzyme-like characteristics consistent with the biochemical model: Stu2 stimulated microtubule growth rates with hyperbolic dependence on tubulin concentration, and the amount of Stu2 on the microtubule end did not vary with tubulin concentration (microtubule growth rate). Complementary measurements of TOG:tubulin binding revealed high affinity (10 nM) and slow dissociation (0.03 s-1). The polymerase and binding measurements can be unified within the biochemical model: Stu2 operates with high efficiency, acting as a tubulin-shuttling antenna on the microtubule end that is primarily limited by the rate of tubulin:TOG association. Our work thus provides a quantitative biochemical mechanism for TOG-based polymerases. That unrelated microtubule and actin polymerases use the same enzyme-like mechanism provides an example of convergent evolution in the cytoskeleton.

cell biology↗

bioRxiv2024

Structures of the multi-domain oxygen sensor DosP: remote control of a c-di-GMP phosphodiesterase by a regulatory PAS domain

The heme-based direct oxygen sensor DosP degrades c-di-GMP, a second messenger nearly unique to bacteria. In stationary phase Escherichia coli, DosP is the most abundant c-di-GMP phosphodiesterase. Ligation of O2 to a heme-binding PAS domain (hPAS) of the protein enhances the phosphodiesterase through an allosteric mechanism that has remained elusive. We determined six structures of full-length DosP in its aerobic or anaerobic conformations, with or without c-di-GMP. DosP is an elongated dimer with the regulatory heme and phosphodiesterase separated by nearly 180 [A]. In the absence of substrate, regardless of the heme status, DosP presents an equilibrium of two distinct conformations. Binding of substrate induces DosP to adopt a single, ON-state or OFF-state conformation depending on its heme status. Structural and biochemical studies of this multi-domain sensor and its mutants provide insights into signal regulation of second-messenger levels.

biochemistry↗

bioRxiv2023

Development of a mutant aerosolized ACE2 that neutralizes SARS-CoV-2 in vivo

The rapid evolution of SARS-CoV-2 variants highlights the need for new therapies to prevent disease spread. SARS-CoV-2, like SARS-CoV-1, uses the human cell surface protein angiotensin-converting enzyme 2 (ACE2) as its native receptor. Here, we design and characterize a mutant ACE2 that enables rapid affinity purification of a dimeric protein by altering the active site to prevent autoproteolytic digestion of a C-terminal His10 epitope tag. In cultured cells, mutant ACE2 competitively inhibits lentiviral vectors pseudotyped with spike from multiple SARS-CoV-2 variants, and infectious SARS-CoV-2. Moreover, the protein can be nebulized and retains virus-binding properties. We developed a system for delivery of aerosolized ACE2 to K18-hACE2 mice and demonstrate protection by our modified ACE2 when delivered as a prophylactic agent. These results show proof-of-concept for an aerosolized delivery method to evaluate anti-SARS-CoV-2 agents in vivo and suggest a new tool in the ongoing fight against SARS-CoV-2 and other ACE2-dependent viruses.

microbiology↗

bioRxiv2020

Functional insights from biophysical study of TREM2 interactions with ApoE and Aβ1-42

INTRODUCTIONTREM2 is an innate immune receptor expressed on myeloid cells including microglia in the brain. How TREM2 engages different ligands remains poorly understood. METHODSWe used comprehensive BLI analysis to investigate the TREM2 interactions with ApoE and monomeric amyloid beta (mA{beta}42). RESULTSTREM2 binding did not depend on ApoE lipidation, and there were only slight differences in affinity observed between ApoE isoforms (E4 > E3 > E2). Surprisingly, disease-linked TREM2 variants within a "basic patch" minimally impact ApoE binding. Instead, TREM2 has a unique hydrophobic surface that can bind to ApoE. This direct engagement requires the hinge region of ApoE. TREM2 directly binds mA{beta}42 and can potently inhibit A{beta}42 polymerization, suggesting a potential mechanism for soluble TREM2 (sTREM2) in preventing AD pathogenesis. DISCUSSIONThese findings demonstrate that TREM2 has at least two separate surfaces to engage ligands and uncovers a potential function for sTREM2 in directly inhibiting A{beta} polymerization.

biophysics↗