The homeodomain of Oct4 is a dimeric binder of methylated CpG elements.

Oct4 is essential to maintain pluripotency and has a pivotal role in establishing the germline. Its DNA-binding POU domain was recently found to bind motifs with methylated CpG elements normally associated with epigenetic silencing. However, the mode of binding and the consequences of this capability has remained unclear. Here, we show that Oct4 binds to a compact palindromic DNA element with a methylated CpG core (CpGpal) in alternative states of pluripotency and during cellular reprogramming towards induced pluripotent stem cells (iPSCs). During cellular reprogramming, typical Oct4 bound enhancers are uniformly demethylated, with the prominent exception of the CpGpal sites where DNA methylation is often maintained. We demonstrate that Oct4 cooperatively binds the CpGpal element as a homodimer, which contrasts with the ectoderm-expressed POU factor Brn2. Indeed, binding to CpGpal is Oct4-specific as other POU factors expressed in somatic cells avoid this element. Binding assays combined with structural analyses and molecular dynamic simulations show that dimeric Oct4-binding to CpGpal is driven by the POU-homeodomain whilst the POU-specific domain is detached from DNA. Collectively, we report that Oct4 exerts parts of its regulatory function in the context of methylated DNA through a DNA recognition mechanism that solely relies on its homeodomain.

OCT4 interprets and enhances nucleosome flexibility.

Pioneer transcription factors are proteins that induce cellular identity transitions by binding to inaccessible regions of DNA in nuclear chromatin. They contribute to chromatin opening and recruit other factors to regulatory DNA elements. The structural features and dynamics modulating their interaction with nucleosomes are still unresolved. From a combination of experiments and molecular simulations, we reveal here how the pioneer factor and master regulator of pluripotency, Oct4, interprets and enhances nucleosome structural flexibility. The magnitude of Oct4's impact on nucleosome dynamics depends on the binding site position and the mobility of the unstructured tails of nucleosomal histone proteins. Oct4 uses both its DNA binding domains to propagate and stabilize open nucleosome conformations, one for specific sequence recognition and the other for nonspecific interactions with nearby regions of DNA. Our findings provide a structural basis for the versatility of transcription factors in engaging with nucleosomes and have implications for understanding how pioneer factors induce chromatin dynamics.

Histone tails cooperate to control the breathing of genomic nucleosomes.

Genomic DNA is packaged in chromatin, a dynamic fiber variable in size and compaction. In chromatin, repeating nucleosome units wrap 145-147 DNA basepairs around histone proteins. Genetic and epigenetic regulation of genes relies on structural transitions in chromatin which are driven by intra- and inter-nucleosome dynamics and modulated by chemical modifications of the unstructured terminal tails of histones. Here we demonstrate how the interplay between histone H3 and H2A tails control ample nucleosome breathing motions. We monitored large openings of two genomic nucleosomes, and only moderate breathing of an engineered nucleosome in atomistic molecular simulations amounting to 24 µs. Transitions between open and closed nucleosome conformations were mediated by the displacement and changes in compaction of the two histone tails. These motions involved changes in the DNA interaction profiles of clusters of epigenetic regulatory aminoacids in the tails. Removing the histone tails resulted in a large increase of the amplitude of nucleosome breathing but did not change the sequence dependent pattern of the motions. Histone tail modulated nucleosome breathing is a key mechanism of chromatin dynamics with important implications for epigenetic regulation.

ArhGEF37 assists dynamin 2 during clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) engages over 30 proteins to secure efficient cargo and membrane uptake. While the function of most core CME components is well established, auxiliary mechanisms crucial for fine-tuning and adaptation remain largely elusive. In this study, we identify ArhGEF37, a currently uncharacterized protein, as a constituent of CME. Structure prediction together with quantitative cellular and biochemical studies present a unique BAR domain and PI(4,5)P2-dependent protein-membrane interactions. Functional characterization yields accumulation of ArhGEF37 at dynamin 2-rich late endocytic sites and increased endocytosis rates in the presence of ArhGEF37. Together, these results introduce ArhGEF37 as a regulatory protein involved in endocytosis.

Chromatosome Structure and Dynamics from Molecular Simulations.

Chromatosomes are fundamental units of chromatin structure that are formed when a linker histone protein binds to a nucleosome. The positioning of the linker histone on the nucleosome influences the packing of chromatin. Recent simulations and experiments have shown that chromatosomes adopt an ensemble of structures that differ in the geometry of the linker histone-nucleosome interaction. In this article we review the application of Brownian, Monte Carlo, and molecular dynamics simulations to predict the structure of linker histone-nucleosome complexes, to study the binding mechanisms involved, and to predict how this binding affects chromatin fiber structure. These simulations have revealed the sensitivityof the chromatosome structure to variations in DNA and linker histone sequence, as well as to posttranslational modifications, thereby explaining the structural variability observed in experiments. We propose that a concerted application of experimental and computational approaches will reveal the determinants of chromatosome structural variability and how it impacts chromatin packing.

Nucleosomal DNA Dynamics Mediate Oct4 Pioneer Factor Binding.

Transcription factor (TF) proteins bind to DNA to regulate gene expression. Normally, accessibility to DNA is required for their function. However, in the nucleus, the DNA is often inaccessible, wrapped around histone proteins in nucleosomes forming the chromatin. Pioneer TFs are thought to induce chromatin opening by recognizing their DNA binding sites on nucleosomes. For example, Oct4, a master regulator and inducer of stem cell pluripotency, binds to DNA in nucleosomes in a sequence-specific manner. Here, we reveal the structural dynamics of nucleosomes that mediate Oct4 binding from molecular dynamics simulations. Nucleosome flexibility and the amplitude of nucleosome motions such as breathing and twisting are enhanced in nucleosomes with multiple TF binding sites. Moreover, the regions around the binding sites display higher local structural flexibility. Probing different structures of Oct4-nucleosome complexes, we show that alternative configurations in which Oct4 recognizes partial binding sites display stable TF-DNA interactions similar to those observed in complexes with free DNA and compatible with the DNA curvature and DNA-histone interactions. Therefore, we propose a structural basis for nucleosome recognition by a pioneer TF that is essential for understanding how chromatin is unraveled during cell fate conversions.

DNA-mediated dimerization on a compact sequence signature controls enhancer engagement and regulation by FOXA1.

FOXA1 is a transcription factor capable to bind silenced chromatin to direct context-dependent cell fate conversion. Here, we demonstrate that a compact palindromic DNA element (termed 'DIV' for its diverging half-sites) induces the homodimerization of FOXA1 with strongly positive cooperativity. Alternative structural models are consistent with either an indirect DNA-mediated cooperativity or a direct protein-protein interaction. The cooperative homodimer formation is strictly constrained by precise half-site spacing. Re-analysis of chromatin immunoprecipitation sequencing data indicates that the DIV is effectively targeted by FOXA1 in the context of chromatin. Reporter assays show that FOXA1-dependent transcriptional activity declines when homodimeric binding is disrupted. In response to phosphatidylinositol-3 kinase inhibition DIV sites pre-bound by FOXA1 such as at the PVT1/MYC locus exhibit a strong increase in accessibility suggesting a role of the DIV configuration in the chromatin closed-open dynamics. Moreover, several disease-associated single nucleotide polymorphisms map to DIV elements and show allelic differences in FOXA1 homodimerization, reporter gene expression and are annotated as quantitative trait loci. This includes the rs541455835 variant at the MAPT locus encoding the Tau protein associated with Parkinson's disease. Collectively, the DIV guides chromatin engagement and regulation by FOXA1 and its perturbation could be linked to disease etiologies.

Changing POU dimerization preferences converts Oct6 into a pluripotency inducer.

The transcription factor Oct4 is a core component of molecular cocktails inducing pluripotent stem cells (iPSCs), while other members of the POU family cannot replace Oct4 with comparable efficiency. Rather, group III POU factors such as Oct6 induce neural lineages. Here, we sought to identify molecular features determining the differential DNA-binding and reprogramming activity of Oct4 and Oct6. In enhancers of pluripotency genes, Oct4 cooperates with Sox2 on heterodimeric SoxOct elements. By re-analyzing ChIP-Seq data and performing dimerization assays, we found that Oct6 homodimerizes on palindromic OctOct more cooperatively and more stably than Oct4. Using structural and biochemical analyses, we identified a single amino acid directing binding to the respective DNA elements. A change in this amino acid decreases the ability of Oct4 to generate iPSCs, while the reverse mutation in Oct6 does not augment its reprogramming activity. Yet, with two additional amino acid exchanges, Oct6 acquires the ability to generate iPSCs and maintain pluripotency. Together, we demonstrate that cell type-specific POU factor function is determined by select residues that affect DNA-dependent dimerization.

Dependence of Chromatosome Structure on Linker Histone Sequence and Posttranslational Modification.

Linker histone (LH) proteins play a key role in higher-order structuring of chromatin for the packing of DNA in eukaryotic cells and in the regulation of genomic function. The common fruit fly (Drosophila melanogaster) has a single somatic isoform of the LH (H1). It is thus a useful model organism for investigating the effects of the LH on nucleosome compaction and the structure of the chromatosome, the complex formed by binding of an LH to a nucleosome. The structural and mechanistic details of how LH proteins bind to nucleosomes are debated. Here, we apply Brownian dynamics simulations to compare the nucleosome binding of the globular domain of D. melanogaster H1 (gH1) and the corresponding chicken (Gallus gallus) LH isoform, gH5, to identify residues in the LH that critically affect the structure of the chromatosome. Moreover, we investigate the effects of posttranslational modifications on the gH1 binding mode. We find that certain single-point mutations and posttranslational modifications of the LH proteins can significantly affect chromatosome structure. These findings indicate that even subtle differences in LH sequence can significantly shift the chromatosome structural ensemble and thus have implications for chromatin structure and transcriptional regulation.

Conformational selection and dynamic adaptation upon linker histone binding to the nucleosome.

Linker histones are essential for DNA compaction in chromatin. They bind to nucleosomes in a 1:1 ratio forming chromatosomes. Alternative configurations have been proposed in which the globular domain of the linker histone H5 (gH5) is positioned either on- or off-dyad between the nucleosomal and linker DNAs. However, the dynamic pathways of chromatosome assembly remain elusive. Here, we studied the conformational plasticity of gH5 in unbound and off-dyad nucleosome-bound forms with classical and accelerated molecular dynamics simulations. We find that the unbound gH5 converts between open and closed conformations, preferring the closed form. However, the open gH5 contributes to a more rigid chromatosome and restricts the motion of the nearby linker DNA through hydrophobic interactions with thymidines. Moreover, the closed gH5 opens and reorients in accelerated simulations of the chromatosome. Brownian dynamics simulations of chromatosome assembly, accounting for a range of amplitudes of nucleosome opening and different nucleosome DNA sequences, support the existence of both on- and off-dyad binding modes of gH5 and reveal alternative, sequence and conformation-dependent chromatosome configurations. Taken together, these findings suggest that the conformational dynamics of linker histones and nucleosomes facilitate alternative chromatosome configurations through an interplay between induced fit and conformational selection.

Ligand tunnels in T. brucei and human CYP51: Insights for parasite-specific drug design.

BACKGROUND: Cytochrome P450 sterol 14α-demethylase (CYP51) is an essential enzyme for sterol biosynthesis and a target for anti-parasitic drug design. However, the design of parasite-specific drugs that inhibit parasitic CYP51 without severe side effects remains challenging. The active site of CYP51 is situated in the interior of the protein. Here, we characterize the potential ligand egress routes and mechanisms in Trypanosoma brucei and human CYP51 enzymes. METHODS: We performed Random Acceleration Molecular Dynamics simulations of the egress of four different ligands from the active site of models of soluble and membrane-bound T. brucei CYP51 and of soluble human CYP51. RESULTS: In the simulations, tunnel 2f, which leads to the membrane, was found to be the predominant ligand egress tunnel for all the ligands studied. Tunnels S, 1 and W, which lead to the cytosol, were also used in T. brucei CYP51, whereas tunnel 1 was the only other tunnel used significantly in human CYP51. The common tunnels found previously in other CYPs were barely used. The ligand egress times were shorter for human than T. brucei CYP51, suggesting lower barriers to ligand passage. Two gating residues, F105 and M460, in T. brucei CYP51 that modulate the opening of tunnels 2f and S were identified. CONCLUSIONS: Although the main egress tunnel was the same, differences in the tunnel-lining residues, ligand passage and tunnel usage were found between T. brucei and human CYP51s. GENERAL SIGNIFICANCE: The results provide a basis for the design of selective anti-parasitic agents targeting the ligand tunnels.

DNA-mediated cooperativity facilitates the co-selection of cryptic enhancer sequences by SOX2 and PAX6 transcription factors.

Sox2 and Pax6 are transcription factors that direct cell fate decision during neurogenesis, yet the mechanism behind how they cooperate on enhancer DNA elements and regulate gene expression is unclear. By systematically interrogating Sox2 and Pax6 interaction on minimal enhancer elements, we found that cooperative DNA recognition relies on combinatorial nucleotide switches and precisely spaced, but cryptic composite DNA motifs. Surprisingly, all tested Sox and Pax paralogs have the capacity to cooperate on such enhancer elements. NMR and molecular modeling reveal very few direct protein-protein interactions between Sox2 and Pax6, suggesting that cooperative binding is mediated by allosteric interactions propagating through DNA structure. Furthermore, we detected and validated several novel sites in the human genome targeted cooperatively by Sox2 and Pax6. Collectively, we demonstrate that Sox-Pax partnerships have the potential to substantially alter DNA target specificities and likely enable the pleiotropic and context-specific action of these cell-lineage specifiers.

Cooperative DNA Recognition Modulated by an Interplay between Protein-Protein Interactions and DNA-Mediated Allostery.

Highly specific transcriptional regulation depends on the cooperative association of transcription factors into enhanceosomes. Usually, their DNA-binding cooperativity originates from either direct interactions or DNA-mediated allostery. Here, we performed unbiased molecular simulations followed by simulations of protein-DNA unbinding and free energy profiling to study the cooperative DNA recognition by OCT4 and SOX2, key components of enhanceosomes in pluripotent cells. We found that SOX2 influences the orientation and dynamics of the DNA-bound configuration of OCT4. In addition SOX2 modifies the unbinding free energy profiles of both DNA-binding domains of OCT4, the POU specific and POU homeodomain, despite interacting directly only with the first. Thus, we demonstrate that the OCT4-SOX2 cooperativity is modulated by an interplay between protein-protein interactions and DNA-mediated allostery. Further, we estimated the change in OCT4-DNA binding free energy due to the cooperativity with SOX2, observed a good agreement with experimental measurements, and found that SOX2 affects the relative DNA-binding strength of the two OCT4 domains. Based on these findings, we propose that available interaction partners in different biological contexts modulate the DNA exploration routes of multi-domain transcription factors such as OCT4. We consider the OCT4-SOX2 cooperativity as a paradigm of how specificity of transcriptional regulation is achieved through concerted modulation of protein-DNA recognition by different types of interactions.

OCT4: dynamic DNA binding pioneers stem cell pluripotency.

OCT4 was discovered more than two decades ago as a transcription factor specific to early embryonic development. Early studies with OCT4 were descriptive and looked at determining the functional roles of OCT4 in the embryo as well as in pluripotent cell lines derived from embryos. Later studies showed that OCT4 was one of the transcription factors in the four-factor cocktail required for reprogramming somatic cells into induced pluripotent stem cells (iPSCs) and that it is the only factor that cannot be substituted in this process by other members of the same protein family. In recent years, OCT4 has emerged as a master regulator of the induction and maintenance of cellular pluripotency, with crucial roles in the early stages of differentiation. Currently, mechanistic studies look at elucidating the molecular details of how OCT4 contributes to establishing selective gene expression programs that define different developmental stages of pluripotent cells. OCT4 belongs to the POU family of proteins, which have two conserved DNA-binding domains connected by a variable linker region. The functions of OCT4 depend on its ability to recognize and bind to DNA regulatory regions alone or in cooperation with other transcription factors and on its capacity to recruit other factors required to regulate the expression of specific sets of genes. Undoubtedly, future iPSC-based applications in regenerative medicine will benefit from understanding how OCT4 functions. Here we provide an integrated view of OCT4 research conducted to date by reviewing the different functional roles for OCT4 and discussing the current progress in understanding their underlying molecular mechanisms. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.

Dynamics of CYP51: implications for function and inhibitor design.

Sterol 14α-demethylase (cytochrome P450 family 51 (CYP51)) is an essential enzyme occurring in all biological kingdoms. In eukaryotes, it is located in the membrane of the endoplasmic reticulum. Selective inhibitors of trypanosomal CYP51s that do not affect the human CYP51 have been discovered in vitro and found to cure acute and chronic mouse Chagas disease without severe side effects in vivo. Crystal structures indicate that CYP51 may be more rigid than most CYPs, and it has been proposed that this property may facilitate antiparasitic drug design. Therefore, to investigate the dynamics of trypanosomal CYP51, we built a model of membrane-bound Trypanosoma brucei CYP51 and then performed molecular dynamics simulations of T. brucei CYP51 in membrane-bound and soluble forms. We compared the dynamics of T. brucei CYP51 with those of human CYP51, CYP2C9, and CYP2E1. In the simulations, the CYP51s display low mobility in the buried active site although overall mobility is similar in all the CYPs studied. The simulations suggest that in CYP51, pathway 2f serves as the major ligand access tunnel, and both pathways 2f (leading to membrane) and S (leading to solvent) can serve as ligand egress tunnels. Compared with the other CYPs, the residues at the entrance of the ligand access tunnels in CYP51 have higher mobility that may be necessary to facilitate the passage of its large sterol ligands. The water (W) tunnel is accessible to solvent during most of the simulations of CYP51, but its width is affected by the conformations of the heme's two propionate groups. These differ from those observed in the other CYPs studied because of differences in their hydrogen-bonding network. Our simulations give insights into the dynamics of CYP51 that complement the available experimental data and have implications for drug design against CYP51 enzymes.

Regulation of the activity of lactate dehydrogenases from four lactic acid bacteria.

Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose 1,6-bisphosphate (FBP), phosphate (Pi), and ionic strength (NaCl concentration) on six LDHs from four LABs studied at pH 6 and pH 7. We found that 1) the extent of activation by FBP (Kact) differs. Lactobacillus plantarum LDH is not regulated by FBP, but the other LDHs are activated with increasing sensitivity in the following order: Enterococcus faecalis LDH2 ≤ Lactococcus lactis LDH2 < E. faecalis LDH1 < L. lactis LDH1 ≤ Streptococcus pyogenes LDH. This trend reflects the electrostatic properties in the allosteric binding site of the LDH enzymes. 2) For L. plantarum, S. pyogenes, and E. faecalis, the effects of Pi are distinguishable from the effect of changing ionic strength by adding NaCl. 3) Addition of Pi inhibits E. faecalis LDH2, whereas in the absence of FBP, Pi is an activator of S. pyogenes LDH, E. faecalis LDH1, and L. lactis LDH1 and LDH2 at pH 6. These effects can be interpreted by considering the computed binding affinities of Pi to the catalytic and allosteric binding sites of the enzymes modeled in protonation states corresponding to pH 6 and pH 7. Overall, the results show a subtle interplay among the effects of Pi, FBP, and pH that results in different regulatory effects on the LDHs of different LABs.

Organism-adapted specificity of the allosteric regulation of pyruvate kinase in lactic acid bacteria.

Pyruvate kinase (PYK) is a critical allosterically regulated enzyme that links glycolysis, the primary energy metabolism, to cellular metabolism. Lactic acid bacteria rely almost exclusively on glycolysis for their energy production under anaerobic conditions, which reinforces the key role of PYK in their metabolism. These organisms are closely related, but have adapted to a huge variety of native environments. They include food-fermenting organisms, important symbionts in the human gut, and antibiotic-resistant pathogens. In contrast to the rather conserved inhibition of PYK by inorganic phosphate, the activation of PYK shows high variability in the type of activating compound between different lactic acid bacteria. System-wide comparative studies of the metabolism of lactic acid bacteria are required to understand the reasons for the diversity of these closely related microorganisms. These require knowledge of the identities of the enzyme modifiers. Here, we predict potential allosteric activators of PYKs from three lactic acid bacteria which are adapted to different native environments. We used protein structure-based molecular modeling and enzyme kinetic modeling to predict and validate potential activators of PYK. Specifically, we compared the electrostatic potential and the binding of phosphate moieties at the allosteric binding sites, and predicted potential allosteric activators by docking. We then made a kinetic model of Lactococcus lactis PYK to relate the activator predictions to the intracellular sugar-phosphate conditions in lactic acid bacteria. This strategy enabled us to predict fructose 1,6-bisphosphate as the sole activator of the Enterococcus faecalis PYK, and to predict that the PYKs from Streptococcus pyogenes and Lactobacillus plantarum show weaker specificity for their allosteric activators, while still having fructose 1,6-bisphosphate play the main activator role in vivo. These differences in the specificity of allosteric activation may reflect adaptation to different environments with different concentrations of activating compounds. The combined computational approach employed can readily be applied to other enzymes.

Structural dynamics in chromatin unraveling by pioneer transcription factors.

Pioneer transcription factors are proteins with a dual function. First, they regulate transcription by binding to nucleosome-free DNA regulatory elements. Second, they bind to DNA while wrapped around histone proteins in the chromatin and mediate chromatin opening. The molecular mechanisms that connect the two functions are yet to be discovered. In recent years, pioneer factors received increased attention mainly because of their crucial role in promoting cell fate transitions that could be used for regenerative therapies. For example, the three factors required to induce pluripotency in somatic cells, Oct4, Sox2, and Klf4 were classified as pioneer factors and studied extensively. With this increased attention, several structures of complexes between pioneer factors and chromatin structural units (nucleosomes) have been resolved experimentally. Furthermore, experimental and computational approaches have been designed to study two unresolved, key scientific questions: First, do pioneer factors induce directly local opening of nucleosomes and chromatin fibers upon binding? And second, how do the unstructured tails of the histones impact the structural dynamics involved in such conformational transitions? Here we review the current knowledge about transcription factor-induced nucleosome dynamics and the role of the histone tails in this process. We discuss what is needed to bridge the gap between the static views obtained from the experimental structures and the key structural dynamic events in chromatin opening. Finally, we propose that integrating nuclear magnetic resonance spectroscopy with molecular dynamics simulations is a powerful approach to studying pioneer factor-mediated dynamics of nucleosomes and perhaps small chromatin fibers using native DNA sequences.

Highly cooperative chimeric super-SOX induces naive pluripotency across species.

Our understanding of pluripotency remains limited: iPSC generation has only been established for a few model species, pluripotent stem cell lines exhibit inconsistent developmental potential, and germline transmission has only been demonstrated for mice and rats. By swapping structural elements between Sox2 and Sox17, we built a chimeric super-SOX factor, Sox2-17, that enhanced iPSC generation in five tested species: mouse, human, cynomolgus monkey, cow, and pig. A swap of alanine to valine at the interface between Sox2 and Oct4 delivered a gain of function by stabilizing Sox2/Oct4 dimerization on DNA, enabling generation of high-quality OSKM iPSCs capable of supporting the development of healthy all-iPSC mice. Sox2/Oct4 dimerization emerged as the core driver of naive pluripotency with its levels diminished upon priming. Transient overexpression of the SK cocktail (Sox+Klf4) restored the dimerization and boosted the developmental potential of pluripotent stem cells across species, providing a universal method for naive reset in mammals.

Conformational diversity and ligand tunnels of mammalian cytochrome P450s.

The mammalian cytochrome P450 (CYP) enzymes play important roles in drug metabolism, steroid biosynthesis, and xenobiotic degradation. The active site of CYPs is buried in the protein and thus the ligands have to enter and exit the active site via ligand tunnels. Conformational changes of flexible parts of the protein usually accompany the entrance and exit of ligands. Comparison of the crystal structures of mammalian CYPs in closed, open, and partially open states reveals that the greatest conformational diversity associated with ligand tunnel opening is in the regions of the B-C and F-G loops. Some CYPs have been observed to adopt different open and closed conformations when bound to different ligands, suggesting that the ligand entrance and exit routes might differ according to the ligand properties. Mammalian CYPs are mostly membrane-bound enzymes, making them difficult to characterize structurally and dynamically. A range of molecular dynamics simulation techniques has been applied to investigate the dynamics and the ligand tunnels of these proteins both in the aqueous environment, and more recently, in lipid bilayers. These simulations not only reveal multiple tunnels through which ligands can pass but also show that different tunnels are preferred by different ligands and that the lipid bilayer can influence the protein dynamics and tunnel opening. The results indicate that not only the active site but also the ligand tunnels can contribute to the different substrate specificity profiles of the mammalian CYPs.