Biology subjects

Ludtke, S.

Publications and source records attributed to Ludtke, S..

4 recordsLinked to original sources

bioRxiv2023

In situ structures of secretins from bacterial type II secretion system reveal their membrane interactions and translocation process

The GspD secretin is the outer membrane channel of the bacterial type II secretion system (T2SS) which secrets diverse effector proteins or toxins that cause severe diseases such as diarrhea and cholera. GspD needs to translocate from the inner to the outer membrane to exert its function, and this process is an essential step for T2SS to assemble. Here, we investigate two types of secretins discovered so far in Escherichia coli, GspD and GspD{beta}, respectively. By electron cryotomography subtomogram averaging, we determine in situ structures of all the key intermediate states of GspD and GspD{beta} in the translocation process, with resolution ranging from 9 [A] to 19 [A]. In our results, GspD and GspD{beta} present entirely different membrane interaction patterns and ways of going across the peptidoglycan layer. We propose two distinct models for the membrane translocation of GspD and GspD{beta}, providing a comprehensive perspective on the inner to outer membrane biogenesis of T2SS secretins.

microbiology↗

bioRxiv2022

Structural basis for tunable affinity and specificity of LxCxEdependentprotein interactions with the retinoblastoma protein family

The retinoblastoma protein (Rb) and its homologs p107 and p130 are critical regulators of gene expression during the cell cycle. Functional inactivation of Rb family proteins leads to loss of cell-cycle control and promotes genome instability and proliferation, which are hallmarks of cancer. Rb proteins share a structural domain, known as the "pocket domain", which mediates association with a large number of cellular proteins. A cleft in the pocket domain binds an LxCxE sequence motif in these proteins, many of which function with Rb proteins to co-regulate transcription during quiescence and G1. Proteins from oncogenic DNA viruses also bind this cleft to inactivate Rb family proteins, and the E7 protein from the human papillomavirus has been used as the primary model for understanding LxCxE motif interactions. Evidence presented previously and here demonstrates that the E7 sequence binds tighter and competes with cellular proteins for the LxCxE site, but the molecular basis for the relatively weak affinities of the cellular proteins has been poorly understood. Here, we present binding data and a series of crystal structures of the p107 pocket domain in complex with LxCxE peptides derived from the transcriptional co-repressor proteins HDAC1, ARI4DA, and EID1. The structures reveal determinants of protein binding beyond the L-C-E core that include the N and C-terminal sides of the LxCxE motif and the x (wild card) residues. Our results explain how differences in these interactions outside the L-C-E core result in weaker affinities of pocket protein for cellular LxCxE proteins compared to E7 and result in differences in Rb and p107 affinities for some LxCxE-containing sequences. Our study provides new insights into how Rb proteins bind their myriad of cell partners and lays the foundation for structure-based design of peptides that inhibit oncogenic viral protein interactions.

biochemistry↗

bioRxiv2021

Cryo-ET distinguishes platelets during pre-acute myeloid leukemia from steady state hematopoiesis

Early diagnosis of acute myeloid leukemia (AML) in the pre-leukemic stage remains a clinical challenge, as pre-leukemic patients show no symptoms, lacking any known morphological or numerical abnormalities in blood cells. Here, we demonstrate that platelets with structurally abnormal mitochondria emerge at the pre-leukemic phase of AML, preceding detectable changes in blood cell counts or detection of leukemic blasts in blood. We visualized frozen-hydrated platelets from mice at different time points during AML development in situ using electron cryo-tomography (cryo-ET) and identified intracellular organelles through an unbiased semi-automatic process followed by quantitative measurement. A large proportion of platelets exhibited changes in the overall shape and depletion of organelles in AML. Notably, 23% of platelets in pre-leukemic cells exhibit abnormal, round mitochondria with unfolded cristae, accompanied by a significant drop in ATP levels and altered expression of metabolism-related gene signatures. Our study demonstrates that detectable structural changes in pre-leukemic platelets may serve as a biomarker for the early diagnosis of AML.

biophysics↗

bioRxiv2020

In situ structure of the AcrAB-TolC efflux pump at subnanometer resolution

In Gram-negative bacteria, tripartite efflux pump AcrAB-TolC plays a prominent role in antibiotic resistance. We have used high resolution cryo-ET to visualize the structure of Escherichia coli AcrAB-TolC at a 7 [A] resolution in intact cells. The resulting structures show the detailed architecture of the assembled complex embedded into cell envelope. Interactions with the inner membrane enable crosstalk between AcrB and TolC through AcrA, suggesting that assembly in the native cellular environment is critical for the pump activation mechanism, where the allosteric activating signal is triggered by the alternate binding of AcrA to the lipid membrane and AcrB porter domain. We establish a platform for high resolution in situ structural studies of bacteria efflux pump, which can yield critical information in understanding complex assemblies function. One sentence summaryA 7 [A] in situ structure of the AcrAB-TolC pump from bacteria by cryo-ET reveals mechanisms for active efflux.

biophysics↗