Biology subjects

Baker, R. W.

Publications and source records attributed to Baker, R. W..

11 recordsLinked to original sources

bioRxiv2025

X-ray crystal structure of the N-terminal domain of Staphylococcus aureus cell-cycle protein GpsB

GpsB is a conserved cell-cycle regulator in the Firmicute clade of Gram-positive bacteria that coordinates multiple aspects of envelope biogenesis. Recent studies demonstrate interactions between GpsB and the key division cytoskeleton FtsZ, suggesting that GpsB links cell division to various aspects of cell envelope biogenesis in Staphylococcus aureus and potentially other Firmicutes. We determined a 1.7 [A] resolution crystal structure of the N-terminal domain of Staphylococcus aureus GpsB, revealing an asymmetric dimer with a bent conformation. This conformation is nearly identical to one of two conformations reported by Sacco, et al., confirming the unique conformation of S. aureus GpsB compared to other gram-positive bacteria. This structural agreement provides strong validation of the S. aureus GpsB fold and supports its proposed role in organizing the cell division machinery.

microbiology↗

bioRxiv2025

CCDC32 collaborates with the membrane to assemble the AP-2 clathrin adaptor complex

Cells have evolved a variety of assembly chaperones to aid in the difficult process of forming macromolecular complexes in a crowded cytoplasm. Assembly of adaptor protein complex 2 (AP-2), the primary cargo adaptor in clathrin-mediated endocytosis, is regulated by the chaperones AAGAB and CCDC32, whose deletion causes loss of all AP-2 subunits in vivo. AAGAB and CCDC32 are thought to act sequentially to assemble the AP-2 tetramer from its constituent heterodimers. However, the molecular requirements and structural consequences of CCDC32 interaction with AP-2 are not yet understood. Here, using in vitro reconstitution and integrative structural analysis, we describe the molecular mechanism of CCDC32-mediated AP-2 assembly. First, CCDC32 interacts with the appendage domain of the AP-2 subunit, using the same binding site as canonical endocytic regulators in addition to a novel, yet highly conserved pocket on . CCDC32 contains cargo sorting motifs normally found in trans-membrane cargo and binds to AP-2 heterodimers using canonical cargo-binding sites. Additionally, two amphipathic helices in CCDC32 bind to the /{sigma}2 heterodimer. Surprisingly, in solution, we find that CCDC32 prevents complex assembly and actively disassembles AP-2 tetramers. Inhibition requires the amphipathic helices of CCDC32, which also mediate binding to PIP2-containing membranes. The presence of PIP2-containing membrane stabilizes the final stages of assembly. We propose that the membrane acts as a molecular switch to release inhibitory interactions, allowing for full complex assembly to proceed. Using cryo-EM, we visualize an assembly intermediate that mimics the conformation of AP-2 found in vesicles, with CCDC32 bound at both cargo binding sites and both membrane-binding sites, suggesting that assembly leads to deposition of active complexes on the plasma membrane.

molecular biology↗

bioRxiv2025

Nickel-NTA lipid-monolayer affinity grids allow for high-resolution structure determination by cryo-EM

Grid preparation is a rate-limiting step in determining high-resolution structures by single particle cryogenic electron microscopy (cryo-EM). Particle interaction with the air-water interface often leads to denaturation, aggregation, or a preferred orientation within the ice. Some samples yield insufficient quantities of particles when using traditional grid making techniques and require the use of solid supports that concentrate samples onto the grid. Recent advances in grid-preparation show that affinity grids are promising tools to selectively concentrate proteins while simultaneously protecting samples from the air-water interface. One such technique utilizes lipid monolayers containing a lipid species with an affinity handle. Some of the first affinity grids used a holey carbon layer coated with nickel nitrilotriacetic acid (Ni-NTA) lipid, which allowed for the binding of proteins bearing the commonly used poly-histidine affinity tag. These studies however used complicated protocols and were conducted before the "resolution revolution" of cryo-EM. Here, we provide a straight-forward preparation method and systematic analysis of Ni-NTA lipid monolayers as a tool for high-resolution single particle cryo-EM. We found that lipid affinity grids concentrate particles away from the air-water interface in thin ice ([~]30 nm). We determined a 2.6 [A] structure of the human nucleosome, showing this method is amenable to high-resolution structure determination. Furthermore, we determined a 3.1 [A] structure of a sub-100 kDa protein demonstrating that this technique is amenable to proteins across biological size ranges. Lipid monolayers are therefore an easily extendable tool for most systems and help alleviate common problems such as low yield, disruption by the air-water interface, and thicker ice.

biochemistry↗

bioRxiv2024

A structure-based mechanism for initiation of AP-3 coated vesicle formation

Adaptor protein complex 3 (AP-3) mediates cargo sorting from endosomes to lysosomes and lysosome-related organelles. Recently, it was shown that AP-3 is in a constitutively open, active conformation compared to the related AP-1 and AP-2 coat complexes, which are inactive until undergoing large conformational changes upon membrane recruitment. How AP-3 is regulated is therefore an open question. To understand the mechanism of AP-3 membrane recruitment and activation, we reconstituted the core of human AP-3 and determined multiple structures in the soluble and membrane-bound states using electron cryo-microscopy (cryo-EM). Similar to yeast AP-3, human AP-3 is in a constitutively open conformation, with the cargo-binding domain of the 3 subunit conformationally free. To reconstitute AP-3 activation by the small GTPase Arf1, we used lipid nanodiscs to build Arf1-AP-3 complexes on membranes and determined three structures that show the stepwise conformational changes required for formation of AP-3 coated vesicles. First, membrane-recruitment is driven by one of two predicted Arf1 binding sites on AP-3. In this conformation, AP-3 is flexibly tethered to the membrane and its cargo binding domain remains conformationally dynamic. Second, cargo binding causes AP-3 to adopt a fixed position and rigidifies the complex, which stabilizes binding for a second Arf1 molecule. Finally, binding of the second Arf1 molecule provides the template for AP-3 dimerization, providing a glimpse into the first step of coat polymerization. We propose coat polymerization only occurs after cargo engagement, thereby linking cargo sorting with assembly of higher order coat structures. Additionally, we provide evidence for two amphipathic helices in AP-3, suggesting that AP-3 contributes to membrane deformation during coat assembly. In total, these data provide evidence for the first stages of AP-3 mediated vesicle coat assembly.

molecular biology↗

bioRxiv2023

Identification of FacZ as a division site placement factor in Staphylococcus aureus

Staphylococcus aureus is a gram-positive pathogen responsible for life-threatening infections that are difficult to treat due to antibiotic resistance. The identification of new vulnerabilities in essential processes like cell envelope biogenesis represents a promising avenue towards the development of anti-staphylococcal therapies that overcome resistance. To this end, we performed cell sorting-based enrichments for S. aureus mutants with defects in envelope integrity and cell division. We identified many known envelope biogenesis factors as well as a large collection of new factors with roles in this process. Mutants inactivated for one of the hits, the uncharacterized SAOUHSC_01855 protein, displayed aberrant membrane invaginations and multiple FtsZ cytokinetic ring structures. This factor is broadly distributed among Firmicutes, and its inactivation in B. subtilis similarly caused division and membrane defects. We therefore renamed the protein FacZ (Firmicute-associated coordinator of Z-rings). In S. aureus, inactivation of the conserved cell division protein GpsB suppressed the division and morphological defects of facZ mutants. Additionally, FacZ and GpsB were found to interact directly in a purified system. Thus, FacZ is a novel antagonist of GpsB function with a conserved role in controlling division site placement in S. aureus and other Firmicutes.

microbiology↗

bioRxiv2023

Lipid nanodiscs as a template for high-resolution cryo-EM structures of peripheral membrane proteins

Peripheral membrane proteins are ubiquitous throughout cell biology and are required for a variety of cellular processes such as signal transduction, membrane trafficking, and autophagy. Transient binding to the membrane has a profound impact on protein function, serving to induce conformational changes and alter biochemical and biophysical parameters by increasing the local concentration of factors and restricting diffusion to two dimensions. Despite the centrality of the membrane in serving as a template for cell biology, there are few reported high-resolution structures of peripheral membrane proteins bound to the membrane. We analyzed the utility of lipid nanodiscs to serve as a template for cryo-EM analysis of peripheral membrane proteins. We tested a variety of nanodiscs and we report a 3.3 [A] structure of the AP2 clathrin adaptor complex bound to a 17-nm nanodisc, with sufficient resolution to visualize a bound lipid head group. Our data demonstrate that lipid nanodiscs are amenable to high-resolution structure determination of peripheral membrane proteins and provide a framework for extending this analysis to other systems.

biochemistry↗

bioRxiv2020

Structural insights into assembly and function of the RSC chromatin remodeling complex

Chromatin remodelers regulate the position and composition of nucleosomes throughout the genome, producing different remodeling outcomes despite a shared underlying mechanism based on a conserved RecA DNA translocase. How this functional diversity is achieved remains unknown despite recent cryo-electron microscopy (cryo-EM) reconstructions of several remodelers, including the yeast RSC complex. To address this, we have focused on a RSC subcomplex comprising its ATPase (Sth1), the essential actin-related proteins (ARPs) Arp7 and Arp9, and the fungal-specific protein Rtt102. Combining cryo-EM and biochemistry of this subcomplex, which exhibits regulation of remodeling by the ARPs, we show that ARP binding induces a helical conformation in the HSA domain of Sth1, which bridges the ATPase domain with the bulk of the complex. Surprisingly, the ARP module is rotated by 120{degrees} in the subcomplex relative to full RSC about a pivot point previously identified as a regulatory hub in Sth1, suggesting that large conformational changes are part of Sth1 regulation and RSC assembly. We also show that an interaction between Sth1 and the nucleosome acidic patch, which appears to be conserved among SWI/SNF remodelers, enhances remodeling. Taken together, our structural data shed light on the assembly and function of the RSC complex.

molecular biology↗

bioRxiv2019

A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex

Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in C. elegans. Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.

cell biology↗

bioRxiv2019

Lis1 promotes the formation of maximally activated cytoplasmic dynein-1 complexes

Cytoplasmic dynein-1 is a molecular motor that drives nearly all minus-end-directed microtubule-based transport in human cells, performing functions ranging from retrograde axonal transport to mitotic spindle assembly1,2. Activated dynein complexes consist of one or two dynein dimers, the dynactin complex, and an \"activating adaptor\", with maximal velocity seen with two dimers present (Fig. 1a)3-6. Little is known about how this massive [~]4MDa complex is assembled. Using purified recombinant human proteins, we uncovered a novel role for the dynein-binding protein, Lis1, in the formation of fully activated dynein complexes containing two dynein dimers. Lis1 is required for maximal velocity of complexes activated by proteins representing three different families of activating adaptors: BicD2, Hook3, and Ninl. Once activated dynein complexes have formed, they do not require the presence of Lis1 for sustained maximal velocity. Using cryo-electron microscopy we show that human Lis1 binds to dynein at two sites on dyneins motor domain, similar to yeast dynein7. We propose that the ability of Lis1 to bind at these sites may function in multiple stages of assembling the motile human dynein/ dynactin/ activating adaptor complex.\n\nO_FIG O_LINKSMALLFIG WIDTH=123 HEIGHT=200 SRC=\"FIGDIR/small/683052v1_fig1.gif\" ALT=\"Figure 1\">\nView larger version (37K):\norg.highwire.dtl.DTLVardef@1acc26corg.highwire.dtl.DTLVardef@1107393org.highwire.dtl.DTLVardef@113eab4org.highwire.dtl.DTLVardef@95f6f4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Lis1 increases microtubule binding and the velocity of activated dynein complexes. a. Schematic of the current model for dynein activation. Dynein is autoinihibited in the Phi conformation, opens and then adopts a parallel conformation that is seen in the activated dynein complex, which includes dynactin and an activating adaptor. Maximally activated dynein contains two dynein dimers (dynein A and B, far right) b. Schematic of the dynein motor domain with AAA+ ATPase domains colored in rainbow, highlighting the two Lis1 binding sites in yeast dynein, \"sitering\" at AAA3/4 and \"sitestalk\" on dyneins stalk, which leads to dyneins microtubule binding domain (MTBD). Lis1 is shown in orange to the right c. Schematic of the activating adaptor constructs used in this study. d. Binding density (mean {+/-} s.e.m. dynein) of dynein alone on microtubules in the absence (white circles) or presence (black circles) of 300 nM Lis1. Data was normalized to a density of 1.0 in the absence of Lis1. Statistical analysis was performed using a two-tailed unpaired t test; ****, p<0.0001; n = 12 replicates for each condition. e. Binding density (mean {+/-} s.e.m) of dynein/ dynactin/ activating adaptor complexes on microtubules in the absence (white circles) or presence (black circles) of 300 nM Lis1. The activating adaptors used are indicated. Data was normalized to a density of 1.0 in the absence of Lis1. Statistical analysis was performed using a two-tailed unpaired t test; ****, p<0.0001; n = 12 replicates for each condition. f. Velocity of dynein/ dynactin/ activating adaptor complexes in the absence (white circles) or presence (black circles) of 300 nM Lis1. The median and interquartile range are shown and the activating adaptors used are indicated. Statistical analysis was performed using a two-tailed Mann-Whitney test; ****, p<0.0001; ns, p=0.3498; n (individual single molecule events) = 506 (BicD2-S no Lis1), 569 (BicD2-S with Lis1), 496 (BicD2-L no Lis1), 505 (BicD2-L with Lis1), 454 (Hook3 no Lis1), 471 (Hook3 with Lis1), 490 (Ninl no Lis1), 582 (Ninl with Lis1). g. Percent processive runs (mean {+/-} s.e.m.) of dynein/ dynactin/ Hook3 complexes in a higher salt buffer (60 mM KOAc versus 30 mM KOAc in our standard motility buffer) in the absence (white circles) or presence (black circles) of 300 nM Lis1. Statistical analysis was performed using a one-way ANOVA and Tukeys multiple comparisons test; ****, p<0.0001; n = 3 replicates per condition. h. Schematic of the peroxisome relocation assay. i. Peroxisome velocity in human U2OS cells with scrambled or Lis1 siRNA knockdown. The median and interquartile range are shown. Statistical analysis was performed using a two-tailed unpaired t test; ***, p=0.0002; n (average peroxisome velocity per cell) = 30 per condition. More than 7 events were measured per cell.\n\nC_FIG

biochemistry↗

bioRxiv2018

Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association

Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. Next, SNAP-25 binds the templated SNAREs to form a partially-zippered SNARE complex. Finally, full zippering displaces Munc18-1. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.

biophysics↗

bioRxiv2018

Repeat-specific functions for the C-terminal domain of RNA polymerase II in budding yeast

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) is required to regulate transcription and to integrate it with other essential cellular processes. In the budding yeast Saccharomyces cerevisiae, the CTD of Rpb1p consists of 26 conserved heptad repeats that are post-translationally modified to orchestrate protein factor binding at different stages of the transcription cycle. A long-standing question in the study of the CTD is if there are any functional differences between the 26 repeats. In this study, we present evidence that repeats of identical sequence have different functions based on their position within the CTD. We assembled plasmids expressing Rpb1p with serine to alanine substitutions in three defined regions of the CTD and measured a range of phenotypes for yeast expressing these constructs. Mutations in the beginning and middle regions of the CTD had drastic, and region-specific effects, while mutating the distal region had no observable phenotype. Further mutational analysis determined that Ser5 within the first region of repeats was solely responsible for the observed growth differences and sequencing fast-growing suppressors allowed us to further define the functional regions of the CTD. This mutational analysis is consistent with current structural models for how the RNAPII holoenzyme and the CTD specifically would reside in complex with Mediator and establishes a foundation for studying regioselective binding along the repetitive RNAPII CTD.

genetics↗