Biology subjects

Hilbert, B. J.

Publications and source records attributed to Hilbert, B. J..

5 recordsLinked to original sources

bioRxiv2020

Viral Packaging ATPases Utilize a Glutamate Switch to Couple ATPase Activity and DNA Translocation

Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in apo, ATP-bound, and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an inactive to an active pose upon ATP binding, and that a residue assigned as the glutamate switch is necessary for regulating the transition. Further, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental data. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the predicted structural coupling from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the {varphi}29 DNA packaging motor. Our work thus establishes a signaling pathway in viral DNA packaging motors that ensures coordination between chemical and mechanical events involved in viral DNA packaging.

biophysics↗

bioRxiv2020

The ΔF508 CFTR defect: molecular mechanism of suppressor mutation V510D and the contribution of transmembrane helix unraveling

Cystic fibrosis (CF) results from mutations within the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a transmembrane chloride channel found on the apical surface of epithelial cells. The most common CF-causing mutation results in a deletion of phenylalanine 508 ({Delta}F508-CFTR), a residue normally found within the NBD1 domain. Loss of F508 causes NBD1 to be less thermodynamically stable and prevents proper tertiary folding of CFTR. As a result, CFTR is not properly trafficked to the cell surface. Recently, progress has been made towards the development of small molecule "correctors" that can restore CFTR tertiary structure and stabilize the channel to overcome the instability inherent in {Delta}F508-CFTR. However, the resultant improvement in channel activity has been modest, and the need for potent correctors remains. To fully inform such efforts, a better understanding of the molecular pathology associated with {Delta}F508-CFTR is required. Here we present a comprehensive study of the impact of F508 deletion on both purified NBD1 and full-length CFTR. Through the use of homology modeling, molecular dynamics simulations, mutational analysis, biochemical, biophysical and functional characterization studies, we obtained insight into how the {Delta}F508 mutation may lead to helical unraveling of transmembrane domains 10 and 11 (TM10, TM11), and how the known suppressor mutations V510D and R1070W, as well as novel second site suppressor mutations (SSSMs) identified in this work, may act to rescue {Delta}F508-CFTR maturation and trafficking.

biochemistry↗

bioRxiv2019

A thermophilic phage uses a small terminase protein with a fixed helix-turn-helix geometry

Tailed bacteriophage use a DNA packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component acts as a molecular matchmaker by recognizing the viral genome as well as the main motor component, the large terminase (TerL). How TerS binds DNA and the TerL protein remains unclear. Here, we identify the TerS protein of the thermophilic bacteriophage P74-26. TerSP76-26 oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. Our cryo-EM structure shows that TerSP76-26 forms a ring with a wide central pore and radially arrayed helix-turn-helix (HTH) domains. These HTH domains, which are thought to bind DNA by wrapping the helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA binding domain imposes strong constraints on how TerSP76-26 can bind DNA. Finally, the TerSP76-26 structure lacks the conserved C-terminal {beta}-barrel domain used by other TerS proteins for binding TerL, suggesting that a well-ordered C-terminal {beta}-barrel domain is not necessary for TerS to carry out its function as a matchmaker.

biochemistry↗

bioRxiv2017

A hyperthermophilic phage decoration protein suggests common evolutionary origin with Herpesvirus Triplex proteins and an anti-CRISPR protein

Virus capsid proteins reproducibly self-assemble into regularly-shaped, stable shells that protect the viral genome from external environmental assaults, while maintaining the high internal pressure of the tightly packaged viral genome. To elucidate how capsids maintain stability under harsh conditions, we investigated the capsid components of a hyperthermophilic virus, phage P74-26. We determined the structure of a capsid protein gp87 and show that it has the same fold as trimeric decoration proteins that enhance the structural stability of capsids in many other phage, despite lacking significant sequence homology. We also find that gp87 is significantly more stable than its mesophilic homologs, reflecting the high temperature environment in which phage P74-26 thrives. Our analysis of the gp87 structure reveals that the core domain of the decoration protein is conserved in trimeric capsid components across numerous dsDNA viruses, including human pathogens such as Herpesviruses. Moreover, this core {beta}-barrel domain is found in the anti-CRISPR protein AcrIIC1, which suggests a mechanism for the evolution of this broad spectrum Cas9 inhibitor. Our work illustrates the principles for increased stability of a thermophilic decoration protein, and extends the evolutionary reach of the core trimeric decoration protein fold.

biochemistry↗

bioRxiv2016

The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the ATPase that powers DNA translocation and an endonuclease that cleaves the concatemeric genome both at initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage is still mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nucleolysis. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of nucleolysis suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid shell during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the procapsid upon completion of packaging unlocks the nuclease domains to cleave DNA.

biochemistry↗