Elicitation of broadly neutralizing antibodies (bnAbs) is a primary HIV vaccine goal. Native-like trimers mimicking virion-associated spikes present nearly all bnAb epitopes and are therefore ...promising vaccine antigens. However, first generation native-like trimers expose epitopes for non-neutralizing antibodies (non-nAbs), which may hinder bnAb induction. We here employ computational and structure-guided design to develop improved native-like trimers that reduce exposure of non-nAb epitopes in the V3-loop and trimer base, minimize both CD4 reactivity and CD4-induced non-nAb epitope exposure, and increase thermal stability while maintaining bnAb antigenicity. In rabbit immunizations with native-like trimers of the 327c isolate, improved trimers suppress elicitation of V3-directed and tier-1 neutralizing antibodies and induce robust autologous tier-2 neutralization, unlike a first-generation trimer. The improved native-like trimers from diverse HIV isolates, and the design methods, have promise to assist in the development of a HIV vaccine.
The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain ...activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.
Substrate-bound structures of AAA+ protein translocases reveal a conserved asymmetric spiral staircase architecture wherein a sequential ATP hydrolysis cycle drives hand-over-hand substrate ...translocation. However, this configuration is unlikely to represent the full conformational landscape of these enzymes, as biochemical studies suggest distinct conformational states depending on the presence or absence of substrate. Here, we used cryo-electron microscopy to determine structures of the
Lon AAA+ protease in the absence and presence of substrate, uncovering the mechanistic basis for two distinct operational modes. In the absence of substrate, Lon adopts a left-handed, "open" spiral organization with autoinhibited proteolytic active sites. Upon the addition of substrate, Lon undergoes a reorganization to assemble an enzymatically active, right-handed "closed" conformer with active protease sites. These findings define the mechanistic principles underlying the operational plasticity required for processing diverse protein substrates.
We present an atomic model of a substrate-bound inner mitochondrial membrane AAA+ quality control protease in yeast, YME1. Our ~3.4-angstrom cryo-electron microscopy structure reveals how the ...adenosine triphosphatases (ATPases) form a closed spiral staircase encircling an unfolded substrate, directing it toward the flat, symmetric protease ring. Three coexisting nucleotide states allosterically induce distinct positioning of tyrosines in the central channel, resulting in substrate engagement and translocation to the negatively charged proteolytic chamber. This tight coordination by a network of conserved residues defines a sequential, around-the-ring adenosine triphosphate hydrolysis cycle that results in stepwise substrate translocation. A hingelike linker accommodates the large-scale nucleotide-driven motions of the ATPase spiral relative to the planar proteolytic base. The translocation mechanism is likely conserved for other AAA+ ATPases.
In vivo, proteins fold and function in a complex environment subject to many stresses that can modulate a protein’s energy landscape. One aspect of the environment pertinent to protein folding is the ...ribosome, since proteins have the opportunity to fold while still bound to the ribosome during translation.We use a combination of force and chemical denaturant (chemomechanical unfolding), as well as point mutations, to characterize the folding mechanism of the src SH3 domain both as a stalled ribosome nascent chain and free in solution. Our results indicate that src SH3 folds through the same pathway on and off the ribosome. Molecular simulations also indicate that the ribosome does not affect the folding pathway for this small protein. Taken together, we conclude that the ribosome does not alter the folding mechanism of this small protein. These results, if general, suggest the ribosome may exert a bigger influence on the folding of multidomain proteins or protein domains that can partially fold before the entire domain sequence is outside the ribosome exit tunnel.
The work presented here sheds light on molecular mechanisms of Lon, an essential AAA+ quality control protease conserved from bacteria to humans.Chapter 1 highlights the disproportionate impact of ...the cryo-electron microscopy (cryo-EM) “resolution revolution” on AAA+ proteins that led to a conserved translocation mechanism for AAA+ proteases belonging to the classical clade. Although x-ray crystallographic methods elucidated structures of isolated domains from various Lon homologs in addition to distinct conformations of multimeric complexes, these results are in direct conflict with structures of substrate-bound AAA+ proteases. As a result, many questions regarding Lon protease structure and function remain unaddressed.In Chapters 2 and 3, we use cryo-EM to determine substrate-free and substrate-bound conformations of bacterial and human Lon proteases. While the substrate-bound ATPase domains in Lon resemble other AAA+ proteins, the subunits of substrate-free Lon assemble into a distinct open lockwasher configuration. These results provide mechanistic insights on substrate engagement and release, protein translocation, and protease activation in Lon, including an additional layer of regulation evolved in human LONP1.Importantly, these structures provide a framework for understanding the molecular underpinnings of CODAS Syndrome in addition to designing novel therapeutic interventions for diseases associated with mitochondrial dysfunction where Lon activity is implicated, including neurodegeneration and cancer.
We present the first atomic model of a substrate-bound inner mitochondrial membrane AAA+ quality control protease, YME1. Our ~3.4 Å cryo-EM structure reveals how the ATPases form a closed spiral ...staircase encircling an unfolded substrate, directing it toward the flat, symmetric protease ring. Importantly, the structure reveals how three coexisting nucleotide states allosterically induce distinct positioning of tyrosines in the central channel, resulting in substrate engagement and translocation to the negatively charged proteolytic chamber. This tight coordination by a network of conserved residues defines a sequential, around-the-ring ATP hydrolysis cycle that results in step-wise substrate translocation. Furthermore, we identify a hinge-like linker that accommodates the large-scale nucleotide-driven motions of the ATPase spiral independently of the contiguous planar proteolytic base. These results define the first molecular mechanism for a mitochondrial inner membrane AAA+ protease and reveal a translocation mechanism likely conserved for other AAA+ ATPases.
To date, immunization studies of rabbits with the BG505 SOSIP.664 HIV envelope glycoprotein trimers have revealed the 241/289 glycan hole as the dominant neutralizing antibody epitope. Here, we ...isolated monoclonal antibodies from a rabbit that did not exhibit glycan hole-dependent autologous serum neutralization. The antibodies did not compete with a previously isolated glycan hole-specific antibody but did compete with N332 glycan supersite broadly neutralizing antibodies. A 3.5-Å cryoEM structure of one of the antibodies in complex with the BG505 SOSIP.v5.2 trimer demonstrated that while the epitope recognized overlapped the N332 glycan supersite by contacting the GDIR motif at the base of V3, primary contacts were located in the variable V1 loop. These data suggest that strain-specific responses to V1 may interfere with broadly neutralizing responses to the N332 glycan supersite and vaccine immunogens may require engineering to minimize these off-target responses or steer them toward a more desirable pathway.
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