Biology subjects

Kuemmel, D.

Publications and source records attributed to Kuemmel, D..

8 recordsLinked to original sources

bioRxiv2026

Molecular basis of TSC complex GAP activity

The tuberous sclerosis complex (TSC) protein complex (TSCC) acts as the GTPase activating protein (GAP) for the small GTPase Rheb, to limit mTORC1 (mechanistic target of rapamycin complex 1) activity and cellular growth. We report the structure of the catalytic transition state complex of TSCC and Rheb. Assembly of two TSC2 subunits containing "asparagine-thumb" GAP domains with two accessory TSC1 subunits is required for function in cells. Catalysis requires the "asparagine-thumb" residue of TSC2 and conserved residues in Rheb that bind TSC2 at multiple interaction sites. Surprisingly, only one TSC2 GAP domain is catalytically competent and interacts with Rheb. This is realized by asymmetric binding of TSC1, which enables activating structural changes in one of the TSC2 subunits and locks the second TSC2 copy in an inactive conformation. We identify TSC2 variants that affect conformational coupling within TSCC and binding to Rheb. The structure thus explains the catalytic mechanism of TSCC and reveals an allosteric role of TSC1 in this process.

biochemistry↗

bioRxiv2025

Structural Basis for a Scaffolding Role of the COM Domain in Nonribosomal Peptide Synthetases

Nonribosomal peptide synthetases (NRPSs) are multi-domain enzymes that catalyze the biosynthesis of therapeutically relevant natural products. Efficient peptide synthesis relies on intricate domain interactions, whose underlying principles remain poorly understood. The communication-mediating (COM) domains facilitate interactions between separate NRPS subunits like other docking domains, however, exhibit distinctive features that are unusual within this family: COM domains co-occur with epimerization (E) domains, are partially embedded within the adjacent condensation (C) domain and can also be found as an internal cis-COM domain with unknown function. We present the first crystal structure of a cis-COM domain within an E-COM-C domain arrangement from modules 4 and 5 of bacitracin synthetase 3 (BacC). The structure reveals a compactly folded COM domain sandwiched between E and C domains, suggesting a role in orienting these domains for efficient peptidyl carrier protein (PCP) shuttling. Through mutational analyses, dipeptide formation assays, and proximity-dependent photo-crosslinking experiments, we investigated both cis- and trans-COM domains and provide evidence supporting a principal role of COM domains as scaffolds of NRPS architecture. Their function as docking domains may be a secondary consequence of their division into separate donor and acceptor parts.

biochemistry↗

bioRxiv2025

Mechanistic Plasticity of the RabGEFs Mon1-Ccz1 and Fuzzy-Inturned

Rab GTPases organize intracellular trafficking and provide identity to organelles. Their spatiotemporal activation by guanine nucleotide exchange factors (GEFs) is tightly controlled to ensure fidelity. Our structural and functional comparison of the tri-longin domain RabGEFs Mon1-Ccz1 and Fuzzy-Inturned reveals the molecular basis for their target specificity. Both complexes rely on a conserved sequence motif of their substrate GTPases for the catalytic mechanism, while secondary interactions allow discrimination between targets. We also find that dimeric Mon1-Ccz1 from fungi and the metazoan homologs with the additional third subunit RMC1/Bulli bind membranes through electrostatic interactions via distinct interfaces. Protein-lipid interaction studies thus reveal a function of RMC1/Bulli in the complex as mediator of membrane recruitment. In the case of Fuzzy-Inturned, reconstitution experiments demonstrate that the BAR (Bin-Amphiphysin-Rvs) domain protein CiBAR1 can support membrane recruitment of the GEF. Collectively, our study demonstrates the molecular basis for the adaptation of TLD-RabGEFs to different cellular functions.

biochemistry↗

bioRxiv2025

A Cysteine-Less and Ultra-Fast Split Intein Rationally Engineered from Being Aggregation-Prone to Highly Efficient in Protein trans-Splicing

Split inteins ligate their fused extein protein sequences while undergoing self-removal. This unique protein trans-splicing reaction has been harnessed for numerous applications in protein modification. However, several purified split intein precursors splice only partially or are entirely inactive for unknown reasons. We have studied the split Aes123 PolB1 intein, which splices only to about 30%. As a rare representative of cysteine-less split inteins, the Aes intein is attractive due to its resistance to oxidative conditions and orthogonality to thiol chemistries. We revealed that the reduced splicing efficiency is caused by the formation of soluble, {beta}-sheet dominated aggregates of the N-terminal precursor. We computationally, biochemically and biophysically characterized the fully active monomeric fraction to identify sequence regions important for aggregation. Guided by a crystal structure we designed stably monomeric mutants with virtually complete splicing activity. The triple mutant CLm intein (Cysteine-Less and monomeric) retained the ultra-fast rate discovered for the wildtype and exhibits superior utility as a thiol-independent protein modification tool. Characterization of two other benchmark split inteins suggests that the discovered aggregation propensity reflects an inherent challenge to keep the split intein precursor in the partially disordered form required to fold with its complementary partner into the active complex.

biochemistry↗

bioRxiv2024

Structure and mechanism of the RalGAP tumor suppressor complex

The RalGAP (GTPase activating protein) complexes are negative regulators of the Ral GTPases and thus crucial components that counteract (oncogenic) Ras signaling. However, no structural information on the architecture of this tumor suppressor complex is available hampering a mechanistic understanding of its functionality. Here, we present a cryo-EM structure of RalGAP that reveals an extended 58 nm tetrameric architecture comprising two heterodimers of the RalGAP and RalGAP{beta} subunits. We show that the catalytic domain of RalGAP requires stabilization by a unique domain of RalGAP{beta}, providing the molecular basis for why RalGAP complexes are obligatory heterodimers. Formation of RalGAP tetramers is not required for activity in vitro, but essential for function of the complex in vivo. Structural analysis of RalGAP subunit variants reported in cancer patients suggests effects on complex formation and thus functional relevance in tumor development, emphasizing the significance of the obtained structural information for medical research.

biochemistry↗

bioRxiv2023

Regulatory sites in the Mon1-Ccz1 complex control Rab5 to Rab7 transition and endosome maturation

Maturation from early to late endosomes depends on the exchange of their marker proteins Rab5 to Rab7. This requires Rab7 activation by its specific guanine nucleotide exchange factor (GEF) Mon1-Ccz1. Efficient GEF activity of this complex on membranes depends on Rab5, thus driving Rab-exchange on endosomes. However, molecular details on the role of Rab5 in Mon1-Ccz1 activation are unclear. Here we identify key features in Mon1 involved in GEF regulation. We show that the intrinsically disordered N-terminal domain of Mon1 autoinhibits Rab5-dependent GEF-activity on membranes. Consequently, Mon1 truncations result in higher GEF activity in vitro, and a shift from Rab5 to more Rab7 positive structures in Drosophila nephrocytes and yeast, suggesting faster endosomal maturation. Using modeling, we further identify a conserved Rab5 binding site in Mon1. Mutations impairing Rab5 interaction result in poor GEF activity on membranes and growth defects in vivo. Our analysis provides a framework to understand the mechanism of Rab-conversion and organelle maturation along the endomembrane system. SummaryTransport of proteins via the endolysosomal pathway requires Rab5 on early endosomes, which is replaced by Rab7 on late endosomes. Here, we identify distinct regulatory sites in the Rab7 activator, the Mon1-Ccz1 complex, shedding light on the regulation of Rab5 to Rab7 transition.

cell biology↗

bioRxiv2022

Targeting of the Mon1-Ccz1 Rab guanine nucleotide exchange factor to distinct organelles by a synergistic protein and lipid code

Activation of the small GTPase Rab7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 (MC1) is a key step in the maturation of endosomes and autophagosomes. This process is tightly regulated and subject to precise spatiotemporal control of MC1 localization. We here identify and characterize an amphipathic helix in Ccz1, which is required for the function of Mon-Ccz1 in autophagy, but not endosomal maturation. Furthermore, our data show that the interaction of the Ccz1 amphipathic helix with lipid packing defects, binding of Mon1 basic patches to positively charged lipids and association of MC1 with recruiter proteins collectively govern membrane recruitment of the complex in a synergistic and redundant manner. The data demonstrate that specific protein and lipid cues convey the differential targeting of MC1 to endosomes and autophagosomes. We reveal the molecular mechanism how MC1 is adapted to recognizes distinct target compartments by exploiting the unique biophysical properties of organelle membranes and thus provide a model how the complex is regulated and activated independently in different functional contexts.

biochemistry↗

bioRxiv2022

Structure of the lysosomal membrane fusion machinery

Lysosomes are of central importance in cellular recycling, nutrient signaling 1,2 and endocytosis, and are tightly connected to autophagy 3 and the invasion of pathogenic bacteria and viruses 1,4. Lysosomal fusion events are fundamental to cell survival and require HOPS, a conserved heterohexameric tethering complex 5,6. HOPS recognizes and binds small membrane-associated GTPases on lysosomes and organelles, and assembles membrane bound SNAREs for fusion 7,8. Through tethering, HOPS brings membranes in close proximity to each other and significantly increases fusion efficacy by catalysing SNARE assembly. Consequently, different HOPS mutations are causative for severe diseases 6. Despite its fundamental cellular duties, it remained speculative how HOPS fulfils its function as high-resolution structural data were unavailable. Here, we used cryo-electron microscopy to reveal the structure of HOPS. In the complex, two central subunits form the backbone and an assembly hub for the functional domains. Two GTPase binding units extend to opposing ends, while the SNARE binding module points to the side, resulting in a triangular shape of the complex. Unlike previously reported, HOPS is surprisingly rigid and extensive flexibility is confined to its extremities. We show that HOPS complex variants with mutations proximal to the backbone can still tether membranes but fail to efficiently promote fusion indicating, that the observed integrity of HOPS is essential to its function. In our model, the core of HOPS acts as a counter bearing between the flexible GTPase binding domains. This positions the SNARE binding module exactly between the GTPase anchored membranes to promote fusion. Our structural and functional analysis reveals the link between the spectacular architecture of HOPS and its mechanism that couples membrane tethering and SNARE assembly, to catalyse lysosomal fusion.

biophysics↗