Peptide neurotoxins are powerful tools for research, diagnosis, and treatment of disease. Limiting broader use, most receptors lack an identified toxin that binds with high affinity and specificity. This paper describes isolation of toxins for one such orphan target, KcsA, a potassium channel that has been fundamental to delineating the structural basis for ion channel function. A phage-display strategy is presented whereby ∼1.5 million novel and natural peptides are fabricated on the scaffold present in ShK, a sea anemone type I (SAK1) toxin stabilized by three disulfide bonds. We describe two toxins selected by sorting on purified KcsA, one novel (Hui1, 34 residues) and one natural (HmK, 35 residues). Hui1 is potent, blocking single KcsA channels in planar lipid bilayers half-maximally (K i ) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a K i of 0.5 nM whereas Shaker, Kv1.2, and Kv1.3 channels are blocked over 200-fold less well. HmK is potent but promiscuous, blocking KcsA-Shaker, Shaker, Kv1.2, and Kv1.3 channels with K i of 1-4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys 21 and Tyr 22 , are essential for highaffinity binding. Unexpectedly, potassium ions traversing the channel from the inside confer voltage sensitivity to the Hui1 off-rate via Arg 23 , indicating that Lys 21 is not in the pore. The 3D structure of Hui1 reveals a SAK1 fold, rationalizes KcsA inhibition, and validates the scaffold-based approach for isolation of highaffinity toxins for orphan receptors.Hui1 toxin | ShK toxin | HmK toxin | sea anemone | NMR

We show here that membrane-tethered toxins facilitate the biophysical study of the roles of toxin residues in K+ channel blockade to reveal two blocking mechanisms in the K+ channel pore. The structure of the sea anemone type I (SAK1) toxin HmK is determined by NMR. T-HmK residues are scanned by point mutation to map the toxin surface, and seven residues are identified to be critical to occlusion of the KcsA channel pore. T-HmK–Lys22 is shown to interact with K+ ions traversing the KcsA pore from the cytoplasm conferring voltage dependence on the toxin off rate, a classic mechanism that we observe as well with HmK in solution and for Kv1.3 channels. In contrast, two related SAK1 toxins, Hui1 and ShK, block KcsA and Kv1.3, respectively, via an arginine rather than the canonical lysine, when tethered and as free peptides.

Amide-bond equilibrium probability density, P eq = exp­(−u) (u, local potential), and associated conformational entropy, S k = −∫P eq (ln P eq) d Ω ln ∫dΩ, are derived for the Rho GTPase binding domain of Plexin-B1 (RBD) as monomer and dimer from 1 μs MD simulations. The objective is to elucidate the effect of dimerization on the dynamic structure of the RBD. Dispersed (peaked) P eq functions indicate “flexibility” (“rigidity”; the respective concepts are used below in this context). The L1 and L3 loops are throughout highly flexible, the L2 loop and the secondary structure elements are generally rigid, and the L4 loop is flexible in the monomer and rigid in the dimer. Overall, many residues are more flexible in the dimer. These features, and their implications, are discussed. Unexpectedly, we find that monomer unit 1 of the dimer (in short, d1) is unusually flexible, whereas monomer unit 2 (in short, d2) is as rigid as the RBD monomer. This is revealed due to their engagement in slow-to-intermediate conformational exchange detected previously by 15N relaxation experiments. Such motions occur with rates on the order of 103–104 s–1; hence, they cannot be completely sampled over the course of 1 μs simulation. However, the extent to which rigid d2 is affected is small enough to enable physically relevant analysis. The entropy difference between d2 and the monomer yields an entropic contribution of −7 ± 0.7 kJ/mol to the free energy of RBD dimerization. In previous work aimed at similar objectives we used 50–100 ns MD simulations. Those results and the present result differ considerably. In summary, bond-vector P eq functions derived directly from long MD simulations are useful descriptors of protein structural dynamics and provide accurate conformational entropy. Within the scope of slow conformational exchange, they can be useful, even in the presence of incomplete sampling.

We report on progress toward improving NMR relaxation analysis in proteins in terms of the slowly relaxing local structure (SRLS) approach by developing a method that combines SRLS with molecular dynamics (MD) simulations. 15 N−H bonds from the Rho GTPase binding domain of plexin-B1 are used as test case. We focus on the locally restricting/ordering potential of mean force (POMF), u(θ,φ), at the N−H site (θ and φ specify the orientation of the N−H bond in the protein). In SRLS, u(θ,φ) is expanded in the basis set of the real linear combinations of the Wigner rotation matrix elements with M = 0, D L,|K| (θ,φ). Because of limited data sensitivity, only the lowest (L = 2) terms are preserved; this potential function is denoted by u (SRLS) . In MD, the force-fieldparametrized POMF is the potential, u (MD) , defined in terms of the probability distribution, P eq (MD) ∝ exp(−u (MD) ). P eq (MD), and subsequently u (MD) , can be derived from the MD trajectory as histograms. One might contemplate utilizing u (MD) instead of u (SRLS) ; however, histograms cannot be used in SRLS analyses. Here, we approximate u(θ,φ) in terms of linear combinations of the D L,|K| functions with L = 1−4 and appropriate symmetry, denoted by u (DLK) , and optimize the latter (via P eq ) against u (MD) . This yields for every N−H bond an analytical ordering potential, u (DLK-BEST) , which exceeds u (SRLS) considerably in accuracy. u (DLK-BEST) can be used fixed in SRLS data fitting, thereby enabling the determination of additional parameters. This yields a substantially improved picture of structural dynamics, which is a significant benefit. The primary achievement of this work is to have employed for the first time MD data to derive a suitable (in terms of composition and symmetry) approximation to the SRLS POMF.

We have developed a new molecular dynamics (MD) based method for describing analytically local potentials at mobile N–H sites in proteins. Here we apply it to the monomer and dimer of the Rho GTPase binding domain (RBD) of the transmembrane receptor plexin-B1 to gain insight into dimerization, which can compete with Rho GTPase binding. In our method, the local potential is given by linear combinations, u (D L,K ), of the real combinations of the Wigner rotation matrix elements, D L,K , with L = 1–4 and appropriate symmetry. The combination that “fits best” the corresponding MD potential of mean force, u (MD), is the potential we are seeking, u (D L,K  – BEST). For practical reasons the fitting process involves probability distributions, P eq ∝ exp­(−u), instead of potentials, u. The symmetry of the potential, u (D L,K ), may be related to the irreducible representations of the D 2h point group. The monomer (dimer) potentials have mostly Ag and B2u (B1u and B2u) symmetry. For the monomer, the associated probability distributions are generally dispersed in space, shallow, and centered at the “reference N–H orientation” (defined in section 3.1. below); for the dimer many are more concentrated, deep and centered away from the “reference N–H orientation”. The u (D L,K ) functions provide a consistent description of the potential energy landscape at protein N–H sites. The L1-loop of the plexin-B1 RBD is not seen in the crystal structure, and many resonances of the L4 loop are missing in the NMR 15N–1H HSQC spectrum of the dimer; we suggest reasons for these features. An allosteric signal transmission pathway was reported previously for the monomer. We find that it has shallow N–H potentials at its ends, which become deeper as one proceeds toward the middle, complementing structurally the previously derived dynamic picture. Prospects of this study include correlating u (D L,K  – BEST) with MD force-fields, and using them without further adjustment in NMR relaxation analysis schemes.