In eukaryotes, the nucleocytoplasmic transport of macromolecules is mainly mediated by soluble nuclear transport receptors of the karyopherin-β superfamily termed importins and exportins. The highly versatile exportin chromosome region maintenance 1 (CRM1) is essential for nuclear depletion of numerous structurally and functionally unrelated protein and ribonucleoprotein cargoes. CRM1 has been shown to adopt a toroidal structure in several functional transport complexes and was thought to maintain this conformation throughout the entire nucleocytoplasmic transport cycle. We solved crystal structures of free CRM1 from the thermophilic eukaryote Chaetomium thermophilum. Surprisingly, unbound CRM1 exhibits an overall extended and pitched superhelical conformation. The two regulatory regions, namely the acidic loop and the C-terminal α-helix, are dramatically repositioned in free CRM1 in comparison with the ternary CRM1-Ran-Snurportin1 export complex. Single-particle EM analysis demonstrates that, in a noncrystalline environment, free CRM1 exists in equilibrium between extended, superhelical and compact, ring-like conformations. Molecular dynamics simulations show that the C-terminal helix plays an important role in regulating the transition from an extended to a compact conformation and reveal how the binding site for nuclear export signals of cargoes is modulated by different CRM1 conformations. Combining these results, we propose a model for the cooperativity of CRM1 export complex assembly involving the long-range allosteric communication between the distant binding sites of GTPbound Ran and cargo.

Proteins carrying nuclear export signals cooperatively assemble with the export factor CRM1 and the effector protein RanGTP. In lower eukaryotes, this cooperativity is coupled to CRM1 conformational changes; however, it is unknown if mammalian CRM1 maintains its compact conformation or shows similar structural flexibility. Here, combinations of small-angle X-ray solution scattering and electron microscopy experiments with molecular dynamics simulations reveal pronounced conformational flexibility in mammalian CRM1 and demonstrate that RanGTP binding induces association of its N- and C-terminal regions to form a toroid structure. The CRM1 toroid is stabilized mainly by local interactions between the terminal regions, rather than by global strain. The CRM1 acidic loop is key in transmitting the effect of this RanGTP-induced global conformational change to the NES-binding cleft by shifting its population to the open state, which displays enhanced cargo affinity. Cooperative CRM1 export complex assembly thus constitutes a highly dynamic process, encompassing an intricate interplay of global and local structural changes.

Ligand-protein binding processes are essential in biological systems. A well-studied system is the binding of cyclic adenosine monophosphate to the cyclic nucleotide binding domain of the bacterial potassium channel MloK1. Strikingly, the measured on-rate for cyclic adenosine monophosphate binding is two orders of magnitude slower than a simple Smoluchowski diffusion model would suggest. To resolve this discrepancy and to characterize the ligand-binding path in structural and energetic terms, we calculated 1100 ligand-binding molecular dynamics trajectories and tested two scenarios: In the first scenario, the ligand transiently binds to the protein surface and then diffuses along the surface into the binding site. In the second scenario, only ligands that reach the protein surface in the vicinity of the binding site proceed into the binding site. Here, a binding funnel, which increasingly confines the translational as well as the rotational degrees of freedom, determines the binding pathways and limits the on-rate. From the simulations, we identified five surface binding states and calculated the rates between these surface binding states, the binding site, and the bulk. We find that the transient binding of the ligands to the surface binding states does not affect the on-rate, such that this effect alone cannot explain the observed low on-rate. Rather, by quantifying the translational and rotational degrees of freedom and by calculating the binding committor, our simulations confirmed the existence of a binding funnel as the main bottleneck. Direct binding via the binding funnel dominates the binding kinetics, and only ∼10% of all ligands proceed via the surface into the binding site. The simulations further predict an on-rate between 15 and 40μs(mol/l), which agrees with the measured on-rate.

Neurofilaments (NFs) are essential building blocks of axonal architecture. Abnormal behavior of these cytostructural elements has been associated with several neuromuscular disorders such as Amyotropic Lateral Sclerosis (ALS). NFs are assembled from three subunits: Low (NFL), Medium (NFM) and Heavy (NFH). These subunits are characterized by a common alpha helical rod domain and carboxyl terminal domains of different lengths specific to each subunit. The tails project from the core of the filament and contain a number of KSP repeat motifs that belongs to the sites for phosphorylation. Especially, the C-terminal tails of NFM and NFH that have relatively longer lengths and higher number of KSP repeats were found to be the key participants of the sidearmmediated interfilament interactions that regulate the axonal diameter. Though it has been established that that the sidearms play a key functional role, little is known about the roles of individual subunits and the effect of phosphorylation on their behavior. Initially, it was believed that the NFH sidearms play a more integral role in determining axonal structure due to the presence of longer polypeptides and relatively higher KSP repeat units. However, recent studies showed that deleting NFH from neurofilaments does not affect axonal diameter, suggesting that NFM may in fact be the key player. In view of this, it is essential to have an understanding of the morphological behavior of the NFM sidearm in response to physiological conditions. In the present study we carried out MD simulations of human and mouse NFM C terminals under different phosphorylation and ionic conditions. The results from these studies provide useful molecular level insight into the structural changes of NFM sidearms in response to phosphorylation, ionic concentrations. The present study reveals sidearm-mediated regulation mechanism of axonal caliber.

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