Biology subjects

Greber, B. J.

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

12 recordsLinked to original sources

bioRxiv2026

Molecular basis of Cyclin B1-CDK1 recruitment by MAD1

The interaction between Cyclin B1-CDK1 and MAD1 recruits kinase activity to the nuclear pore complex and kinetochore corona to sustain spindle assembly checkpoint (SAC) signaling. Here, we characterize a short linear motif (SLiM) within MAD1 that mediates its interaction with Cyclin B1. This SLiM peptide is specific for B-type cyclins and lacks the canonical "RxL" cyclin-binding consensus sequence, suggesting a distinct mode of cyclin recognition. Using a combination of deep mutational scanning, structural analysis by cryogenic electron microscopy, and binding affinity measurements, we define the molecular basis of the Cyclin B1-MAD1 interaction. These approaches reveal that a short MAD1 peptide binds as an -helix to the RxL-binding hydrophobic patch of Cyclin B1. The interaction employs hydrophobic contacts that overlap with those used by canonical RxL motifs, but is distinguished by additional contacts and sequence features that confer preferential recognition by B-type cyclins while excluding other cyclin types. We also show that the MAD1 motif is sufficient to confer B-type cyclin-specific substrate phosphorylation in mitotic cells. Together, these findings reveal a non-canonical, B-type cyclin-specific helical binding mode that ensures selective Cyclin B1-CDK1 recruitment at two critical stages of SAC signaling, and suggest potential strategies for selective therapeutic targeting of this interaction.

molecular biology↗

bioRxiv2026

Cryo-tides: synthetic peptides for cryo-EM grid preparation of multi-subunit protein complexes sensitive to the air-water interface

Cryogenic electron microscopy (cryo-EM) has enabled rapid progress in structural biology throughout the last decade. However, cryo-EM sample preparation still represents a major bottleneck in high-resolution structure determination. Many proteins and macromolecular complexes interact with the air-water interface during sample preparation, which can cause protein denaturation, complex disassembly, and preferred particle orientation. Development of methods to alleviate these issues is an active field of investigation. However, a universally applicable solution to this problem has not been found so far. Here, we describe the simple and cost-effective usage of synthetic peptides with defined amino acid sequence to protect cryo-EM samples from complex disassembly and reduce preferred orientation. We show benefits during cryo-EM grid preparation of a large E3 ubiquitin ligase termed the anaphase-promoting complex (APC/C), and the small CDK11:cyclin L:SAP30BP complex.

biochemistry↗

bioRxiv2026

Cryo-EM structures of the CDK11-cyclin L-SAP30BP complex reveal mechanisms of CDK11 regulation

The cyclin-dependent kinase CDK11 functions in transcription, mitotic progression, and mRNA splicing. Specifically, spliceosome activation during the B to Bact transition depends on phosphorylation of the U2 snRNP component SF3B1 by the CDK11-cyclin L-SAP30BP complex. Here, we present the structure of this spliceosome-activating CDK-cyclin complex, determined by cryogenic electron microscopy at 2.3 [A] resolution. Our structure and biochemical experiments show that SAP30BP forms extensive interactions with cyclin L2, thereby stabilising it, and forms critical interactions with the C-terminal kinase lobe of CDK11 that promote complex assembly. Destabilisation of cyclin L2 in the absence of SAP30BP suggests that these principles are applicable to all CDK11-cyclin L complexes. Furthermore, we identify a pseudo-substrate sequence near the CDK11 C-terminus and provide evidence for a role of this segment in CDK11 auto-regulation. Finally, the structure of the CDK11-cyclin L2-SAP30BP complex bound to the clinical high-affinity CDK11 inhibitor OTS964 and a comparison to OTS964-bound off-target complexes provide insight into the mechanism of OTS964 selectivity and specificity.

molecular biology↗

bioRxiv2024

High-throughput discovery and deep characterization of cyclin-CDK docking motifs

Cyclin-CDKs are master regulators of cell division. In addition to directly activating the CDK, the cyclin subunit regulates CDK specificity by binding short peptide "docking" motifs in CDK substrates. Here, we measure the relative binding strength of [~]100,000 peptides to 11 human cyclins from five cyclin families (D, E, A, B and F). Using a quantitative intracellular binding assay and large-scale tiled peptide screening, we identified a range of non-canonical binders that unveil a broader than anticipated repertoire of cyclin docking motif types. Structural and saturation mutagenesis studies revealed distinct binding modes and sequence features that govern motif recognition, binding strength, and cyclin preference. Docking motifs vary from highly selective to pan-cyclin, thereby fine-tuning the timing of CDK phosphorylation during cell cycle progression. Overall, these findings provide an unprecedented depth of understanding about the rules encoding specificity and affinity within a group of related but distinct protein domains.

molecular biology↗

bioRxiv2024

Sub-3 A resolution protein structure determination by single-particle cryo-EM at 100 keV

Cryo-electron microscopy (cryo-EM) has revolutionized structural biology by providing high-resolution insights into biological macromolecules. Here, we present sub-3 [A] resolution structures determined using the 100 keV Tundra cryogenic transmission electron microscope (cryo-TEM), equipped with the newly developed Falcon C direct electron detector (DED). Our results demonstrate that this lower voltage microscope, when combined with advanced electron optics and detectors, can achieve high-resolution reconstructions that were previously only attainable with higher voltage systems. The implementation of an extreme-brightness field emission gun (XFEG) and an SP-TWIN objective lens significantly enhanced the spatial and temporal coherence of the system. Furthermore, the semi-automated sample loader minimized contamination and drift, allowing extended data collection sessions without manual intervention. The high detective quantum efficiency (DQE) of Falcon C further improved the signal-to-noise ratio, which is critical for achieving high-resolution structures. We validated the performance of this microscope by determining the structures of various biological samples, including apoferritin, T20S proteasome, GABAA receptor, haemoglobin, and human transthyretin ranging in size from 440 kDa to 50 kDa. The highest resolutions achieved were 2.1 [A] for apoferritin, 2.7 [A] for the 20S proteasome, 2.8 [A] for the GABAA receptor, 5.0 [A] for haemoglobin, and 3.5 [A] for transthyretin. We also explored a larger specimen, a 3.9 MDa Adeno-associated virus (AAV9) capsid and resolved it a 2.8 [A]. This work highlights the potential of 100 keV TEMs to make high-resolution cryo-EM more accessible to the structural biology community. Furthermore, it sets a precedent for the use of lower voltage TEMs in routine cryo-EM studies, not only for screening grids for single particle analysis but also for achieving high-resolution structures of protein samples.

biophysics↗

bioRxiv2023

Structural and quantum chemical basis for OCP-mediated quenching of phycobilisomes

Cyanobacteria employ large antenna complexes called phycobilisomes (PBS) for light harvesting. However, intense light triggers non-photochemical quenching, where the Orange Carotenoid Protein (OCP) binds to PBS, dissipating excess energy as heat. The mechanism of efficiently transferring energy from phycocyanobilins in PBS to canthaxanthin in OCP remains insufficiently understood. Using advanced cryogenic-electron microscopy, we unveiled the OCP-PBS complex structure at 1.6-2.1 [A] resolution, showcasing its inherent flexibility. Employing multiscale quantum chemistry, we disclosed the quenching mechanism. Identifying key protein residues, we clarified how canthaxanthins transition dipole moment in its lowest-energy dark state becomes large enough for efficient energy transfer from phycocyanobilins. Our energy transfer model offers a detailed understanding of the atomic determinants of light harvesting regulation and antenna architecture in cyanobacteria. One sentence summaryHigh-resolution cryo-EM structure of the OCP-PBS complex reveals intrinsic motions and enables the atomic simulation of the quenching mechanism

biophysics↗

bioRxiv2023

High-resolution cryo-electron microscopy of the human CDK-activating kinase for structure-based drug design

Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to macromolecules refractory to crystallisation has been hampered by the often-limiting resolution and throughput of cryogenic electron microscopy (cryo-EM). Here, we use high-resolution cryo-EM to determine structures of the CDK-activating kinase, a master regulator of cell growth and division, in its free and nucleotide-bound states and in complex with 14 inhibitors at up to 1.8 [A] resolution. Our structures provide detailed insight into inhibitor interactions and networks of water molecules in the active site of cyclin- dependent kinase 7. Our data support a previously proposed mechanism contributing to inhibitor selectivity, thereby providing the basis for rational design of next-generation therapeutics. Additionally, our results establish a methodological framework for the use of high-resolution cryo-EM in structure-based drug design.

molecular biology↗

bioRxiv2021

Structures of the Cyanobacterial Phycobilisome

The phycobilisome is an elaborate antenna that is responsible for light-harvesting in cyanobacteria and red-algae. This large macromolecular complex captures incident sunlight and transfers the energy via a network of pigment molecules called bilins to the photosynthetic reaction centers. The phycobilisome of the model organism Synechocystis PCC 6803 consists of a core to which six rods are attached but its detailed molecular architecture and regulation in response to environmental conditions is not well understood. Here we present cryo-electron microscopy structures of the 6.2 MDa phycobilisome from Synechocystis PCC 6803 resolved at 2.1 [A] (rods) to 2.7 [A] (core), revealing three distinct conformations, two previously unknown. We found that two of the rods are mobile and can switch conformation within the complex, revealing a layer of regulation not described previously. In addition, we found a novel linker protein in the structure, that may represent a long-sought subunit that tethers the phycobilisome to the thylakoid membrane. Finally, we show how excitation energy is transferred within the phycobilisome and correlate our structures with known spectroscopic properties. Together, our results provide detailed insights into the biophysical underpinnings of cyanobacterial light harvesting and lay the foundation for bioengineering of future phycobilisome variants and artificial light harvesting systems.

biochemistry↗

bioRxiv2021

Structure of the Quenched Cyanobacterial OCP-Phycobilisome Complex

Photoprotection is an essential mechanism in photosynthetic organisms to balance the harvesting of light energy against the risks of photodamage. In cyanobacteria, photoprotective non-photochemical quenching relies on the interaction between a photoreceptor, the Orange Carotenoid Protein (OCP), and the antenna, the phycobilisome (PBS). Here we report the first structure of the OCP-PBS complex at 2.7 [A] overall resolution obtained by cryo-electron microscopy. The structure shows that the 6.2 MDa PBS is quenched by four 34 kDa OCP organized as two dimers. The complex also reveals that the structure of the active form of the OCP is drastically different than its resting, non-quenching form, with an [~]60 [A] displacement of its regulatory domain. These results provide a high-resolution blueprint of the structural basis of the protective quenching of excess excitation energy that enables cyanobacteria to harvest light energy and fix CO2 across environmentally diverse and dynamic surface of our planet.

biochemistry↗

bioRxiv2021

Structural transitions in the GTP cap visualized by cryo-EM of catalytically inactive microtubules

Microtubules (MTs) are polymers of /{beta}-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes, and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here we use cryo-EM and TIRF microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in -tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice, but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wildtype GDP-MTs. End binding proteins of the EB family have the ability to compact both mutant GTP-lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wildtype MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these first high-resolution structures of truly GTP-bound MTs add mechanistic insight to our understanding of MT dynamic instability. Significance StatementMicrotubules (MTs) are non-equilibrium polymers that switch between states of growth and shrinkage. This property is critical for their function and is a consequence of GTP hydrolysis in the MT. The structure of the stable GTP part of the MT (the GTP cap) has previously been inferred from MTs polymerized with non-hydrolyzable GTP analogs. Here, we report the first high-resolution structures of MTs truly containing GTP, polymerized from mutated, hydrolysis-deficient tubulins. We find that GTP-MTs have an "expanded lattice" and a "closed seam", structural characteristics possibly responsible for stabilizing the GTP cap. These results provide new insight into the structural transitions at growing MT ends, furthering our understanding of the bistable nature of MTs.

biophysics↗

bioRxiv2020

The cryo-electron microscopy structure of the human CDK-activating kinase

The human CDK-activating kinase (CAK), a complex composed of cyclin dependent kinase (CDK) 7, cyclin H, and MAT1, is a critical regulator of transcription initiation and the cell cycle. It acts by phosphorylating the C-terminal heptapeptide repeat domain of the RNA polymerase II subunit Rpb1, which is an important regulatory event in transcription initiation by Pol II, and it phosphorylates the regulatory T-loop of CDKs that control cell-cycle progression. Here, we have determined the three-dimensional structure of the catalytic module of human CAK, revealing the structural basis of its assembly and providing insight into CDK7 activation in this context. The unique third component of the complex, MAT1, substantially extends the interaction interface between CDK7 and cyclin H, explaining its role as a CAK assembly factor, and it forms interactions with the CDK7 T-loop, which may contribute to enhancing CAK activity. We have also determined the structure of the CAK in complex with the covalently bound inhibitor THZ1 in order to provide insight into the binding of inhibitors at the CDK7 active site and aid in the rational design of therapeutic compounds. SignificanceControl of gene expression and the cell cycle is critical for appropriate cell growth and timely cell division. Failure of the mechanisms regulating these processes can result in proliferative diseases. A molecular complex termed the CDK activating kinase (CAK) impinges on both of these regulatory networks in human cells and is thus a possible drug target for treatment of cancer. Here, we use cryo-electron microscopy to describe the detailed molecular structure of the human CAK, revealing its architecture and the interactions between its regulatory elements. Additionally, we have obtained the structure of the CAK in complex with a small-molecule inhibitor.

biochemistry↗

bioRxiv2019

Architecture of the chromatin remodeler RSC and insights into its nucleosome engagement

Eukaryotic DNA is packaged into nucleosome arrays, which are repositioned by chromatin remodeling complexes to control DNA accessibility1,2. The Saccharomyces cerevisiae RSC (Remodeling the Structure of Chromatin) complex, a member of the SWI/SNF chromatin remodeler family, plays critical roles in genome maintenance, transcription, and DNA repair2-4. Here we report cryo-electron microscopy (cryo-EM) and crosslinking mass spectrometry (CLMS) studies of yeast RSC complex and show that RSC is composed of a rigid tripartite core and two flexible lobes. The core structure is scaffolded by an asymmetric Rsc8 dimer and built with the evolutionarily conserved subunits Sfh1, Rsc6, Rsc9 and Sth1. The flexible ATPase lobe, composed of helicase subunit Sth1, Arp7, Arp9 and Rtt102, is anchored through the interactions between the N-terminus of Sth1 and the core. Our cryo-EM analysis also shows that in addition to the expected nucleosome-Sth1 interactions, RSC engages histones and nucleosomal DNA through one arm of the core structure, composed of Rsc8 SWRIM domains, Sfh1 and Npl6. Our findings provide structural insights into the conserved assembly process for all members of the SWI/SNF family of remodelers, and illustrate how RSC selects, engages, and remodels nucleosomes.

molecular biology↗