Biology subjects

Lasham, J.

Publications and source records attributed to Lasham, J..

6 recordsLinked to original sources

bioRxiv2026

Conformational dynamics of ND2 antiporter-like subunit in intact and truncated complex I

Respiratory complex I is a large redox-driven proton pump and contributes to the proton motive force required for ATP generation in mitochondria and bacteria. It has been extensively studied with structural and biochemical approaches leading to enhanced understanding of its three- dimensional architecture. Despite significant progress in deciphering its catalytic function, the redox-coupled proton pumping mechanism remains unclear and extensively debated. The three membrane-bound antiporter-like subunits ND2, ND4 and ND5 are responsible for pumping protons from the negatively charged side (N-side) of the membrane to the positively charged side (P-side). Emerging evidence supports the role of ND5 subunit in releasing pumped protons to the P-side of the membrane, but the question whether ND2 and ND4 also contribute to proton release remains unanswered. Here, we performed atomistic molecular dynamics (MD) simulations of an intact and a truncated model of complex I in which ND5 and ND4 subunits are removed in-silico. The simulation data reveals a previously unobserved conformational change in the P-side region of ND2 subunit that occurs much more easily in a truncated model of complex I, as also estimated based on free energy calculations. The conformational change, in part triggered by the artificial exposure of ND2 subunit to lipid bilayer, results in a local hydration of the region, similar to what has been observed in recent cryo-EM structure of plant CI:CIII2 supercomplex. We discuss the functional relevance of observed conformational transitions and identify residues that can be manipulated with biochemical approaches to shed light on proton pumping by complex I.

biophysics↗

bioRxiv2026

Fast prediction of acidic amino acid sidechain conformations for cryo-EM modeling

Cryogenic-electron microscopy (cryo-EM) has revolutionized the field of protein structural biology. The structures of large membrane proteins are now routinely determined by cryo-EM to near atomic resolution. However, in the medium resolution range of cryo-EM maps (>[~]2 [A]), negatively charged sidechains of acidic residues are not well-resolved due to the negative electrostatic potential of the region. This may lead to incorrect sidechain models for residues like glutamic acid or aspartic acid that are central for proton transfer activity in various respiratory and photosynthetic enzymes. We previously proposed that the acidic residues with weak or non-existent cryo-EM density can be modeled to represent their low proton affinity conformations. Here, we tested this hypothesis on a larger data set of acidic amino acid residues in two high-resolution respiratory complex I structures. By using faster sidechain modeling and proton affinity prediction tools, we created a workflow that generates sidechain conformations of selected amino acid residues. We validated the sidechain conformation predictions by Q-score analysis and atomistic molecular dynamics simulations in different charged states. The proposed workflow provides a way to rapidly obtain sidechain conformations of acidic residues with weak cryo-EM densities and can be integrated into the existing cryo-EM modeling pipelines to speed up sidechain rotamer prediction.

biophysics↗

bioRxiv2023

Role of protonation states in stability of molecular dynamics simulations of high-resolution membrane protein structures

Classical molecular dynamics (MD) simulations provide unmatched spatial and time resolution of protein structure and function. However, accuracy of MD simulations often depends on the quality of force field parameters and the time scale of sampling. Another limitation of conventional MD simulations is that the protonation states of titratable amino acid residues remain fixed during simulations, even though protonation state changes coupled to conformational dynamics are central to protein function. Due to the uncertainty in selecting protonation states, classical MD simulations are sometimes performed with all amino acids modeled in their standard charged states at pH 7. Here we performed and analyzed classical MD simulations on high-resolution cryo-EM structures of two membrane proteins that transfer protons by catalyzing protonation/deprotonation reactions. In simulations performed with amino acids modeled in their standard protonation state the structure diverges far from its starting conformation. In comparison, MD simulations performed with pre-determined protonation states of amino acid residues reproduce the structural conformation, protein hydration, and protein-water and protein-protein interactions of the structure much better. The results suggest it is crucial to perform basic protonation state calculations, especially on structures where protonation changes play an important functional role, prior to launching any MD simulations. Furthermore, the combined approach of protonation state prediction and MD simulations can provide valuable information on the charge states of amino acids in the cryo-EM sample. Even though accurate prediction of protonation states currently remains a challenge, we introduce an approach of combining pKa prediction with cryo-EM density map analysis that helps in improving not only the protonation state predictions, but also the atomic modeling of density data.

biophysics↗

bioRxiv2022

Nanobody engineering for SARS-CoV-2 neutralization and detection

In response to the ongoing SARS-CoV-2 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain antibody fragments, nanobodies. Here, we explore the potential of nanobody engineering in the development of antivirals and diagnostic tools. Through fusion of nanobody domains that target distinct binding sites, we engineered multimodular nanobody constructs that neutralize wild-type SARS-CoV-2 and the Alpha and Delta variants with high potency, with IC50 values up to 50 pM. However, we observed a limitation in the efficacy of multimodular nanobodies against the Beta (B.1.351) and Omicron variants (B.1.1.529), underlining the importance of accounting for viral evolution in the design of biologics. To further explore the applications of nanobody engineering in outbreak management, we present a novel detection assay, based on fusions of nanobodies with fragments of NanoLuc luciferase that can detect sub-nanomolar quantities of the SARS-CoV-2 spike protein in a single step. Our work showcases the potential of nanobody engineering to combat emerging infectious disease.

microbiology↗

bioRxiv2022

Tunnel dynamics of quinone derivatives and its coupling to protein conformational rearrangements in respiratory complex I

Respiratory complex I in mitochondria and bacteria catalyzes the transfer of electrons from NADH to quinone (Q). The free energy available from the reaction is used to pump protons and to establish a membrane proton electrochemical gradient, which drives ATP synthesis. Even though several high-resolution structures of complex I have been resolved, how Q reduction is linked with proton pumping, remains unknown. Here, microsecond long molecular dynamics (MD) simulations were performed on Yarrowia lipolytica complex I structures where Q molecules have been resolved in the ~30 [A] long Q tunnel. MD simulations of several different redox/protonation states of Q reveal the coupling between the Q dynamics and the restructuring of conserved loops and ion pairs. Oxidized quinone stabilizes towards the N2 FeS cluster, a binding mode not previously described in Yarrowia lipolytica complex I structures. On the other hand, reduced (and protonated) species tend to diffuse towards the Q binding sites closer to the tunnel entrance. Mechanistic and physiological relevance of these results are discussed.

biophysics↗

bioRxiv2021

High-resolution structure and dynamics of mitochondrial complex I - insights into the proton pumping mechanism

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a 1 MDa membrane protein complex with a central role in energy metabolism. Redox-driven proton translocation by complex I contributes substantially to the proton motive force that drives ATP synthase. Several structures of complex I from bacteria and mitochondria have been determined but its catalytic mechanism has remained controversial. We here present the cryo-EM structure of complex I from Yarrowia lipolytica at 2.1 [A] resolution, which reveals the positions of more than 1600 protein-bound water molecules, of which [~]100 are located in putative proton translocation pathways. Another structure of the same complex under steady-state activity conditions at 3.4 [A] resolution indicates conformational transitions that we associate with proton injection into the central hydrophilic axis. By combining high-resolution structural data with site-directed mutagenesis and large-scale molecular dynamics simulations, we define details of the proton translocation pathways, and offer new insights into the redox-coupled proton pumping mechanism of complex I.

biochemistry↗