Biology subjects

Lamers, M. H.

Publications and source records attributed to Lamers, M. H..

8 recordsLinked to original sources

bioRxiv2026

Cryo-EM structure of MutL-activated MutH

Strand discrimination after mismatch recognition is the second essential step that is unique to DNA mismatch repair. MutH is a latent endonuclease that cuts the newly synthesized strand at hemi-methylated GATC sites upon activation by MutL, which acts as a signaling protein between mismatch recognition by MutS and strand discrimination by MutH. While much is known about the recognition of mismatches by MutS, little is known about the activation of strand incision by MutH. Here we present the cryo-EM structure of MutL bound to MutH on a hemi-methylated GATC site at 2.8 [A] resolution. MutL binds MutH at the back of the protein, 25 [A] away from the active site. The structure reveals that activation occurs through an allosteric pathway that acts through a push- and-pull mechanism that releases an inhibitory loop from the DNA minor groove enabling histidine 112 to pull the DNA backbone closer into the active site where a lysine 116 positions a water molecule for the nucleophilic attack at the scissile bond 5 to the GATC sequence. Thus, our structure reveals how MutL couples mismatch recognition by MutS to daughter strand discrimination by MutH.

biochemistry↗

bioRxiv2025

Structural and functional analysis of the Mycobacterium tuberculosis MmpS5L5 efflux pump presages a pathway to increased bedaquiline resistance

Bedaquiline, an antitubercular drug that targets ATP-synthase, is a key component of a new oral drug regimen that has revolutionized the treatment of multi drug resistant tuberculosis. Clinical bedaquiline resistance in Mycobacterium tuberculosis has rapidly emerged, primarily due to mutations in the transcriptional repressor, Rv0678 that result in upregulation of the Resistance-Nodulation-Division (RND) efflux pump MmpS5/MmpL5 (MmpS5L5). Here, to understand how MmpS5L5 effluxes bedaquiline, we determined the structure of the MmpS5L5 complex using cryo-electron microscopy, revealing a novel trimeric architecture distinct from the canonical tripartite RND efflux pumps of Gram-negative bacteria. Structure prediction modelling in conjunction with functional genetic analysis indicates that it uses a periplasmic coiled-coil tube to transport molecules across the cell wall. Structure-guided genetic approaches identify MmpL5 mutations that alter bedaquiline transport; these mutations converge on a region in MmpL5 located in the lower portion of the periplasmic cavity, proximal to the outer leaflet of the inner membrane, suggesting a route for bedaquiline entry into the pump. While currently known clinical resistance to bedaquiline is due to pump upregulation, our findings that several MmpL5 variants increase bedaquiline efflux may presage the emergence of additional modes of clinical resistance. Significance StatementResistance to bedaquiline, a cornerstone drug for treating multidrug-resistant tuberculosis, is rapidly emerging due to mutations that upregulate expression of the MmpS5L5 efflux pump. Here, we reveal the cryo-EM structure of this pump, showing a novel trimeric architecture and a unique -helical coiled-coil tube for drug transport. Structure-guided genetic analysis identifies MmpL5 variants that further increase bedaquiline efflux, suggesting potential resistance mechanisms beyond pump upregulation.

microbiology↗

bioRxiv2025

Identification of determinants of high-fidelity DNA synthesis in M. smegmatis DnaE1 through in silico and in vivo approaches

Drug resistance in Mycobacterium tuberculosis presents a major challenge in tuberculosis treatment, highlighting the need to understand the underlying mechanisms. DNA replication plays an important role in the acquisition of drug resistance and the expression of the DNA polymerase DnaE2 during adverse conditions has been associated with increased mutation rates. Here we investigate the functional differences between the high fidelity replicative DNA polymerase DnaE1 and the predicted error-prone DNA polymerase DnaE2, focusing on which amino acid changes affect polymerase fidelity. For this we identify potential fidelity-altering positions using a two-entropies sequence analysis combined with experimental validation to test whether changes of these positions affect the mutation rates. We find that a double mutation in the palm domain of M. smegmatis DnaE1: D431S/R432D, increases mutation frequencies both in vivo and in vitro. The location of these two residues adjacent to the DNA backbone of the template strand suggests that the amino acid change results in a looser grip on the DNA, allowing for the incorporation of incorrect nucleotides. These insights improve our understanding of the mechanisms underlying drug resistance in M. tuberculosis and could help in the development of future strategies to combat it.

molecular biology↗

bioRxiv2025

PyVADesign: a Python-based cloning tool for one-step generation of large mutant libraries

MotivationThe generation and analysis of diverse mutants of a protein is a powerful tool for understanding protein function. However, generating such mutants can be time-consuming, while the commercial option of buying a series of mutant plasmids can be expensive. In contrast, the insertion of a synthesized double stranded DNA (dsDNA) fragment into a plasmid is a fast and low-cost method to generate a large library of mutants with one or more point mutations, insertions, or deletions. ResultsTo aid in the design of these DNA fragments we have developed PyVADesign: a Python package that makes the design and ordering of dsDNA fragments straight-forward and cost-effective. In PyVADesign, the mutations of interest are clustered in different cloning groups for efficient exchange into the target plasmid. Additionally, primers that prepare the target plasmid for insertion of the dsDNA fragment, as well as primers for sequencing, are automatically designed within the same program. AvailabilityPyVADesign is open source and available at https://github.com/CDDLeiden/PyVADesign/. Contactgerard@lacdr.leidenuniv.nl

bioinformatics↗

bioRxiv2024

Promiscuous NAD-dependent dehydrogenases enable efficient bacterial growth on the PET monomer ethylene glycol

Ethylene glycol is widely used as antifreeze agent and monomer of the ubiquitous plastic PET (polyethylene terephthalate). Its global production amounts to more than 50 million tons per year, and it constitutes an environmental pollutant of increasing concern. Although it is generally accepted that bacteria oxidize ethylene glycol to use it as growth substrate, the enzymes involved in this process are not well understood. Here we show that the soil bacterium Paracoccus denitrificans is able to assimilate ethylene glycol efficiently via NAD-dependent alcohol and aldehyde dehydrogenases. Using comparative proteome analysis, we identify a previously unknown gene cluster that is strongly expressed in the presence of ethylene glycol. We report the kinetic parameters and cryo-EM structures of EtgB and EtgA, the key enzymes encoded by this etg gene cluster. These novel biocatalysts pave the way for more efficient biotechnological conversion of ethylene glycol. We furthermore show that the transcriptional activator EtgR controls expression of the etg gene cluster. Directed evolution of P. denitrificans on ethylene glycol results in faster growing strains, which is enabled by increased activities of EtgB and EtgA. Bioinformatic analysis reveals that the etg gene cluster and variants thereof are widely distributed among Proteobacteria, suggesting a previously underappreciated role of NAD-dependent dehydrogenases in microbial ethylene glycol assimilation.

microbiology↗

bioRxiv2023

RecA-NT homology motif in ImuB is essential for mycobacterial ImuA'-ImuB protein interaction and mutasome function

The mycobacterial mutasome - minimally comprising ImuA, ImuB, and DnaE2 proteins - has been implicated in DNA damage-induced mutagenesis in Mycobacterium tuberculosis. ImuB, predicted to enable mutasome function via its interaction with the {beta} clamp, is a catalytically inactive member of the Y-family of DNA polymerases. Like other members of the Y family, ImuB features a recently identified amino acid motif with homology to the RecA-N-terminus (RecA-NT). In RecA, the motif mediates oligomerization of RecA monomers into RecA filaments. Given the role of ImuB in the mycobacterial mutasome, we hypothesized that the ImuB RecA-NT motif might mediate its interaction with ImuA, a RecA homolog of unknown function. To investigate this possibility, we constructed a panel of imuB alleles in which RecA-NT was removed, or mutated. Results from microbiological and biochemical assays indicate that RecA-NT is critical for the interaction of ImuB with ImuA. A region downstream of RecA-NT (ImuB-C) also appears to stabilize the ImuB-ImuA interaction, but its removal does not prevent complex formation. In contrast, replacing two key hydrophobic residues of RecA-NT, L378 and V383, is sufficient to disrupt ImuA-ImuB interaction. To our knowledge, this constitutes the first experimental evidence showing the role of the RecA-NT motif in mediating the interaction between a Y-family member and a RecA homolog.

microbiology↗

bioRxiv2021

Cryo-EM structures reveal how ATP and DNA binding in MutS coordinate the sequential steps of DNA mismatch repair

DNA mismatch repair detects and removes mismatches from DNA reducing the error rate of DNA replication a 100-1000 fold. The MutS protein is one of the key players that scans for mismatches and coordinates the repair cascade. During this, MutS undergoes multiple conformational changes that initiate the subsequent steps, in response to ATP binding, hydrolysis, and release. How ATP induces the different conformations in MutS is not well understood. Here we present four cryo-EM structures of Escherichia coli MutS at sequential stages of the ATP hydrolysis cycle. These structures reveal how ATP binding and hydrolysis induces a closing and opening of the MutS dimer, respectively. Additional biophysical analysis furthermore explains how DNA binding modulates the ATPase cycle by preventing hydrolysis during scanning and mismatch binding, while preventing ADP release in the sliding clamp state. Nucleotide release is achieved when MutS encounters single stranded DNA that is produced during the removal of the daughter strand. This way, the combination of the ATP binding and hydrolysis and its modulation by DNA enable MutS to adopt different conformations needed to coordinate the sequential steps of the mismatch repair cascade.

biochemistry↗

bioRxiv2019

Longitudinal cell division is associated with single mutations in the FtsZ-recruiting SsgB in Streptomyces

In most bacteria, cell division begins with the polymerization of the GTPase FtsZ at the mid-cell, which recruits the division machinery to initiate cell constriction. In the filamentous bacterium Streptomyces, cell division is positively controlled by SsgB, which recruits FtsZ to the future septum sites and promotes Z-ring formation. Here we show via site-saturated mutagenesis that various amino acid substitutions in the highly conserved SsgB protein result in the production of ectopically placed septa, that sever spores diagonally or along the long axis, perpendicular to the division plane. Ectopic septa were especially prominent when cells expressed SsgB variants with substitutions in residue E120. Biochemical analysis of SsgB variant E120G revealed that its interaction with - and polymerization of - FtsZ had been maintained. The crystal structure of S. coelicolor SsgB was resolved and the position of residue E120 suggests its requirement for maintaining the proper angle of helix 3, thus providing a likely explanation for the aberrant septa formed in SsgB E120 substitution mutants. Taken together, our work presents the first example of longitudinal division in a free living bacterium, which is explained entirely by changes in the FtsZ-recruiting protein SsgB.

developmental biology↗