Nathan S. Astrof

Molecular conformation of a peptide fragment of transthyretin in an amyloid fibril

The molecular conformation of peptide fragment 105–115 of transthyretin, TTR(105–115), previously shown to form amyloid fibrils in vitro, has been determined by magic-angle spinning solid-state NMR spectroscopy. 13C and 15N linewidth measurements indicate that TTR(105–115) forms a highly ordered structure with each amino acid in a unique environment. 2D 13C-13C and 15N-13C-13C chemical shift correlation experiments, performed on three fibril samples uniformly 13C,15N-labeled in consecutive stretches of 4 aa, allowed the complete sequence-specific backbone and side-chain 13C and 15N resonance assignments to be obtained for residues 105–114. Analysis of the 15N, 13CO, 13Cα, and 13Cβ chemical shifts allowed quantitative predictions to be made for the backbone torsion angles φ and ψ. Furthermore, four backbone 13C–15N distances were determined in two selectively 13C,15N-labeled fibril samples by using rotational-echo double-resonance NMR. The results show that TTR(105–115) adopts an extended β-strand conformation that is similar to that found in the native protein except for substantial differences in the vicinity of the proline residue.

AL-57, a ligand-mimetic antibody to integrin LFA-1, reveals chemokine-induced affinity up-regulation in lymphocytes

Affinity of integrin lymphocyte function-associated antigen 1 (LFA-1) is enhanced by conformational changes from the low-affinity closed form to the high-affinity (HA) open form of the ligand-binding inserted (I) domain as shown by work with purified I domains. However, affinity up-regulation of LFA-1 on the cell surface by physiological agonists such as chemokines has yet to be demonstrated by monovalent reagents. We characterize a mAb, AL-57 (activated LFA-1 clone 57), that has been developed by phage display that selectively targets the HA open conformation of the LFA-1 I domain. AL-57 discriminates among low-affinity, intermediate-affinity, and HA states of LFA-1. Furthermore, AL-57 functions as a ligand mimetic that binds only upon activation and requires Mg2+ for binding. Compared with the natural ligand intercellular adhesion molecule-1, AL-57 shows a tighter binding to the open I domain and a 250-fold slower off rate. Monovalent Fab AL-57 demonstrates affinity increases on a subset (≈10%) of lymphocyte cell surface LFA-1 molecules upon stimulation with CXCL-12 (CXC chemokine ligand 12). Affinity up-regulation correlates with global conformational changes of LFA-1 to the extended form. Affinity increase stimulated by CXCL-12 is transient and peaks 2 to 5 min after stimulation.

Sevoflurane binds and allosterically blocks integrin lymphocyte function-associated antigen-1.

Background:Volatile anesthetics have been shown to modify immune cell functions via several mechanisms, some of which have been only partially elucidated. We demonstrated that isoflurane inhibits primary leukocyte integrin lymphocyte function-associated antigen-1 (LFA-1) by binding to the allosteric cavity critical for conformational activation to its high-affinity form. It remains to be determined whether the allosteric inhibition of LFA-1 by isoflurane can be generalized to other anesthetics such as sevoflurane. Methods:The effects of sevoflurane on the ability of LFA-1 to bind to its counter-ligand, intercellular adhesion molecule-1, was studied in leukocytes by flow cytometry. To examine whether sevoflurane acts directly on LFA-1, we measured ligand-binding using beads coated with purified LFA-1 protein. To distinguish between competitive versus allosteric inhibition, we analyzed the effects of sevoflurane on both wild-type and mutant-locked high-affinity LFA-1. One-way analysis of variance was employed for statistical analysis of the data. Nuclear magnetic resonance spectroscopy was used to identify sevoflurane binding site(s). Results:Sevoflurane at clinically relevant concentrations inhibited the ligand-binding function of LFA-1 in leukocytes as well as in cell-free assays (P < 0.05). Sevoflurane blocked wild-type but not locked high-affinity LFA-1, thereby demonstrating an allosteric mode of inhibition. Nuclear magnetic resonance spectroscopy revealed that sevoflurane bound to the allosteric cavity, to which LFA-1 allosteric antagonists and isoflurane also bind. Conclusions:This study suggests that sevoflurane also blocks the activation-dependent conformational changes of LFA-1 to the high-affinity form. The allosteric mode of action exemplified by sevoflurane and isoflurane via LFA-1 might represent one of the underlying mechanisms of anesthetic-mediated immunomodulation.

Crystal structure of isoflurane bound to integrin LFA-1 supports a unified mechanism of volatile anesthetic action in the immune and central nervous systems.

Volatile anesthetics (VAs)’ such as isoflurane’ induce a general anesthetic state by binding to specific targets (i.e.’ ion channels) in the central nervous system (CNS). Simultaneously’ VAs modulate immune functions’ possibly via direct interaction with alternative targets on leukocytes. One such target’ the integrin lymphocyte function‐associated antigen‐1 (LFA‐1)’ has been shown previously to be inhibited by isoflurane. A better understanding of the mechanism by which isoflurane alters protein function requires the detailed information about the drug‐protein interaction at an atomic level. Here’ we describe the crystal structure of the LFA‐1 ligand‐binding domain (I domain) in complex with isoflurane at 1.6 A. We discovered that isoflurane binds to an allosteric cavity previously implicated as critical for the transition of LFA‐1 from the low‐ to the high‐affinity state. The isoflurane binding site in the I domain involves an array of amphiphilic interactions’ thereby resembling a “common anesthetic binding motif” previously predicted for authentic VA binding sites. These results suggest that the allosteric modulation of protein function by isoflurane’ as demonstrated for the integrin LFA‐1’ might represent a unified mechanism shared by the interactions of volatile anesthetics with targets in the CNS.— Zhang, H., Astrof, N. S., Liu, J.‐H., Wang, J.‐H., Shimaoka, M. Crystal structure of isoflurane bound to integrin LFA‐1 supports a unified mechanism of volatile anesthetic action in the immune and central nervous systems. FASEB J. 23, 2735‐2740 (2009)

The volatile anesthetic isoflurane perturbs conformational activation of integrin LFA-1 by binding to the allosteric regulatory cavity

The molecular and structural basis of anesthetic interactions with conformations and functionalities of cell surface receptors remains to be elucidated. We have demonstrated that the widely used volatile anesthetic isoflurane blocks the activation‐dependent conformational conversion of integrin lymphocyte function associated antigen‐1 (LFA‐1), the major leukocyte cell adhesion molecule, to a high‐affinity configuration. Perturbation of LFA‐1 activation by isoflurane at clinically relevant concentrations leads to the inhibition of T‐cell interactions with target cells as well as ligand‐triggered intracellular signaling. Nuclear magnetic resonance spectroscopy reveals that isoflurane binds within a cavity in the LFA‐1 ligand‐binding domain, which is a previously identified drug‐binding site for allosteric small‐molecule antagonists that stabilize LFA‐1 in a low‐affinity conformation. These results provide a potential mechanism for the immunomodulatory properties of isoflurane.— Yuki, K., Astrof, N. S., Bracken, C., Yoo, R., Silkworth, W., Soriano, S. G., Shimaoka, M. The volatile anesthetic isoflurane perturbs conformational activation of integrin LFA‐1 by binding to the allosteric regulatory cavity. FASEB J. 22, 4109–4116 (2008)

CO_H(N)CACB experiments for assigning backbone resonances in 13C/15N-labeled proteins.

A triple resonance NMR experiment, denoted CO_H(N)CACB, correlates1HN and 13CO spins with the13Cα and13Cβ spins of adjacent amino acids. Thepulse sequence is an ‘out-and-back’ design that starts with1HN magnetization and transfers coherence viathe 15N spin simultaneously to the 13CO and13Cα spins, followed by transfer to the13Cβ spin. Two versions of the sequence arepresented: one in which the 13CO spins are frequency labeledduring an incremented t1 evolution period prior to transfer ofmagnetization from the 13Cα to the13Cβ resonances, and one in which the13CO spins are frequency labeled in a constant-time mannerduring the coherence transfer to and from the13Cβ resonances. Because 13COand 15N chemical shifts are largely uncorrelated, thetechnique will be especially useful when degeneracy in the1HN-15N chemical shifts hindersresonance assignment. The CO_H(N)CACB experiment is demonstrated usinguniformly 13C/15N-labeled ubiquitin.

NMR Spectroscopy for Studying Integrin Antagonists

The expansion of the modern pharmacopeia is driven by advances in structural genomics that enable the detailed understanding of drug – protein interactions necessary for the identification of novel compounds as well as improved variants of existing drugs. Our research has centered on understanding the mechanism by which a critical class of transmembrane receptor proteins, the integrins, become activated in healthy and disease states. An important component of this research has been in identifying novel inhibitors of integrin function and their mechanism of action. As we describe in the following sections, nuclear magnetic resonance spectroscopy (NMR) has been an essential tool in advances in this area, and is the major focus of this review.