Local Ordering at Mobile Sites in Proteins: Combining Perspectives from NMR Relaxation and Molecular Dynamics

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. 15N–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, DL,|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-field-parametrized POMF is the potential, u(MD), defined in terms of the probability distribution, Peq(MD) ∝ exp­(−u(MD)). Peq(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 DL,|K| functions with L = 1–4 and appropriate symmetry, denoted by u(DLK), and optimize the latter (via Peq) 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

Tethered peptide neurotoxins display two blocking mechanisms in the K+ channel pore as do their untethered analogs.

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

Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display.

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 (Ki) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a Ki 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 Ki of 1-4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys21 and Tyr22, are essential for high-affinity binding. Unexpectedly, potassium ions traversing the channel from the inside confer voltage sensitivity to the Hui1 off-rate via Arg23, indicating that Lys21 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 high-affinity toxins for orphan receptors

Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display.

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 (Ki) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a Ki 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 Ki of 1-4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys21 and Tyr22, are essential for high-affinity binding. Unexpectedly, potassium ions traversing the channel from the inside confer voltage sensitivity to the Hui1 off-rate via Arg23, indicating that Lys21 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 high-affinity toxins for orphan receptors

Ctr1 Intracellular Loop Is Involved in the Copper Transfer Mechanism to the Atox1 Metallochaperone

Understanding the human copper cycle is essential to understand the role of metals in promoting neurological diseases and disorders. One of the cycles controlling the cellular concentration and distribution of copper involves the copper transporter, Ctr1; the metallochaperone, Atox1; and the ATP7B transporter. It has been shown that the C-terminus of Ctr1, specifically the last three amino acids, HCH, is involved in both copper coordination and the transfer mechanism to Atox1. In contrast, the role of the intracellular loop of Ctr1, which is an additional intracellular segment of Ctr1, in facilitating the copper transfer mechanism has not been investigated yet. Here, we combine various biophysical methods to explore the interaction between this Ctr1 segment and metallochaperone Atox1 and clearly demonstrate that the Ctr1 intracellular loop (1) can coordinate Cu­(I) via interactions with the side chains of one histidine and two methionine residues and (2) closely interacts with the Atox1 metallochaperone. Our findings are another important step in elucidating the mechanistic details of the eukaryotic copper cycle

Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

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 high-affinity 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 high-affinity toxins for orphan receptors

Correction to Thiolate Spin Population of Type I Copper in Azurin Derived from ³³S Hyperfine Coupling

Correction to Thiolate Spin Population of Type I Copper in Azurin Derived from ³³S Hyperfine Couplin