Biology subjects

Kaelber, J.

Publications and source records attributed to Kaelber, J..

6 recordsLinked to original sources

bioRxiv2025

Insights into the AAV packaging mechanism: Cryo-EM Structure of the AAV2 Rep-Capsid Packaging Complex

The Adeno-associated virus (AAV) has become the most used viral vector for gene therapy applications to treat monogenic diseases, with over 250 clinical trials and six FDA-approved biologics. AAV has a single-stranded DNA genome encapsulated in a 60-subunit icosahedral protein shell. Assembly of empty capsids occurs in the nucleus, and in a subsequent step, the genome is packaged by the motor activity of the AAV Rep proteins. The translocation of ssDNA has been suggested to occur through one of the twelve channels at the fivefold symmetry axis. While there is substantial evidence that Rep proteins directly interact with the capsid, the specific molecular determinants, stoichiometry, and translocation mechanism remain unknown. To understand how Rep proteins assemble on the capsid, we examined Rep-capsid complexes using cryo-electron microscopy with single-particle reconstruction. Our results show that Rep proteins can assemble into the capsid fivefold pore as either pentameric or hexameric rings. The different ring complexes dock into the pore similarly, using the post-sensor1 {beta}-hairpin motif (pos1{beta}h2) as a capsid interaction module. This interaction induces the folding of a segment in the capsid HI loop, resulting in an expansion of the pos1{beta}h2 {beta}-sheet. Our structures show that any of the AAV Rep proteins can interact with the capsid, and this mode of interaction may be a conserved mechanism across all parvoviruses. Collectively, our results provide insights into the mechanism of AAV genome encapsidation and could inform strategies to improve recombinant AAV packaging efficiency.

biochemistry↗

bioRxiv2024

Structural basis of long-range transcription-translation coupling

Structures recently have been reported of molecular assemblies that mediate transcription-translation coupling in Escherichia coli. In these molecular assemblies, termed "coupled transcription-translation complexes" or "TTC-B", RNA polymerase (RNAP) directly interacts with the ribosome, the transcription elongation factor NusG or its paralog RfaH forms a bridge between RNAP and ribosome, and the transcription elongation factor NusA optionally forms a second bridge between RNAP and ribosome. Here, we report structures of coupled transcription-translation complexes having mRNA spacers between RNAP and ribosome longer than the maximum-length mRNA spacer compatible with formation of TTC-B. The results define a new class of coupled transcription-translation complex, termed "TTC-LC," where "LC" denotes "long-range coupling." TTC-LC differs from TTC-B by a [~]60{degrees} rotation and [~]70 [A] translation of RNAP relative to ribosome, resulting in loss of direct interactions between RNAP and ribosome and creation of a [~]70 [A] gap between RNAP and ribosome. TTC-LC accommodates long mRNA spacers by looping out mRNA from the gap between RNAP and ribosome. We present evidence that TTC-LC is an intermediate in assembling and disassembling TTC-B, mediating pre-TTC-B transcription-translation coupling before a ribosome catches up to RNAP, and mediating post-TTC-B transcription-translation coupling after a ribosome stops moving and RNAP continues moving. We show that TTC-B, but not TTC-LC, is severely defective in intrinsic, RNA-hairpin-dependent termination, and that both TTC-B and TTC-LC are severely defective in Rho-dependent termination.

molecular biology↗

bioRxiv2021

Near-atomic resolution Cryo-EM structure of Mayaro virus identifies key structural determinants of alphavirus particle formation

Mayaro virus (MAYV) is an arthritis-inducing alphavirus circulating in the Americas, with potential to rapidly emerge in new geographical regions and populated environments. Intraparticle heterogeneity has typically limited atomic resolution structures of alphavirus virions, while imposing icosahedral symmetry in data processing prevents characterization of non-icosahedral features. Here, we report a near-atomic resolution cryo-EM structure of the MAYV E1-E2-E3-CP subunit by addressing deviations from icosahedral symmetry within each virus particle. We identified amino acid contacts at E1 protein interfaces forming the icosahedral lattice and investigated their effect on MAYV growth through site-directed mutagenesis. Further, mutation of a short stretch of conserved residues in E2 subdomain D, near an unidentified "pocket factor" including E2Y358, significantly reduced MAYV growth and provides strong evidence that this unknown factor influences assembly. Further, a symmetry-free reconstruction revealed the MAYV virion is not strictly icosahedral, suggesting defects in global symmetry may be a feature of the virus particle budding process. Our study provides insights into alphavirus assembly and suggests a common path in the formation of spherical, enveloped viruses, leading to particle imperfections.

microbiology↗

bioRxiv2020

High yield preparation of outer-membrane protein efflux pumps by in vitro refolding is concentration dependent

Overexpression of tripartite efflux pump systems in gram-negative bacteria are a principal component of antibiotic resistance. High-yield purification of the outer membrane component of these systems will enable biochemical and structural interrogation of their mechanisms of action and allow testing of compounds that target them. However, preparation of these proteins is typically hampered by low yields requiring laborious large-scale efforts. If refolding conditions can be found, refolding these proteins from inclusion bodies can lead to increased yields as compared to membrane isolations. Here, we develop a concentration-dependent folding protocol for refolding TolC, the outer membrane component of the antibiotic efflux pump from Escherichia coli. We show that by our method of re-folding, homotrimeric TolC remains folded in SDS-PAGE, retains binding to an endogenous ligand, and recapitulates the known crystal structure by single particle cryoEM analysis. We find that a key factor in successful re-folding is a concentration dependence of TolC oligomerization. We extended the scheme to CmeC, a homologous protein from Campylobacter jejuni, and find that concentration-dependent oligomerization is a general feature of these systems. Because outer-membrane efflux pump components are ubiquitous across gram-negative species, we anticipate that incorporating a concentration step in re-folding protocols will promote correct refolding allowing for reliable, high-yield preparation of this family of proteins.

biophysics↗

bioRxiv2020

Structural basis of transcription-translation coupling

In bacteria, transcription and translation are coupled processes, in which movement of RNA polymerase (RNAP) synthesizing mRNA is coordinated with movement of the first ribosome translating mRNA. Coupling is modulated by the transcription factors NusG--which is thought to bridge RNAP and ribosome--and NusA. Here, we report cryo-EM structures of Escherichia coli transcription-translation complexes (TTCs) containing different-length mRNA spacers between RNAP and the ribosome active-center P-site. Structures of TTCs containing short spacers show a state incompatible with NusG bridging and NusA binding (TTC-A; previously termed "expressome"). Structures of TTCs containing longer spacers reveal a new state compatible with NusG bridging and NusA binding (TTC-B) and reveal how NusG bridges and NusA binds. We propose that TTC-B mediates NusG- and NusA-dependent transcription-translation coupling. One Sentence SummaryCryo-EM defines states that mediate NusG- and NusA-dependent transcription-translation coupling in bacteria

molecular biology↗

bioRxiv2019

Structural basis of Q-dependent antitermination

Lambdoid bacteriophage Q protein mediates the switch from middle to late bacteriophage gene expression by enabling RNA polymerase (RNAP) to read through transcription terminators preceding bacteriophage late genes. Q loads onto RNAP engaged in promoter-proximal pausing at a Q binding element (QBE) and an adjacent sigma-dependent pause element (SDPE) to yield a \"Q-loading complex,\" and Q subsequently translocates with RNAP as a pausing-deficient, termination-deficient \"Q-loaded complex.\" Here, we report high-resolution structures of four states on the pathway of antitermination by Q from bacteriophage 21 (Q21): Q21, the Q21-QBE complex, the Q21-loading complex, and the Q21-loaded complex. The results show that Q21 forms a torus--a \"nozzle\"--that narrows and extends the RNAP RNA-exit channel, extruding single-stranded RNA and preventing formation of pause and terminator hairpins.\n\nOne Sentence SummaryQ forms a \"nozzle\" that narrows the RNA polymerase RNA-exit channel and extrudes ssRNA, preventing formation of RNA hairpins.

molecular biology↗