An inducible amphipathic α-helix mediates subcellular targeting and membrane binding of RPE65.

RPE65 retinol isomerase is an indispensable player in the visual cycle between the vertebrate retina and RPE. Although membrane association is critical for RPE65 function, its mechanism is not clear. Residues 107-125 are believed to interact with membranes but are unresolved in all RPE65 crystal structures, whereas palmitoylation at C112 also plays a role. We report the mechanism of membrane recognition and binding by RPE65. Binding of aa107-125 synthetic peptide with membrane-mimicking micellar surfaces induces transition from unstructured loop to amphipathic α-helical (AH) structure but this transition is automatic in the C112-palmitoylated peptide. We demonstrate that the AH significantly affects palmitoylation level, membrane association, and isomerization activity of RPE65. Furthermore, aa107-125 functions as a membrane sensor and the AH as a membrane-targeting motif. Molecular dynamic simulations clearly show AH-membrane insertion, supporting our experimental findings. Collectively, these studies allow us to propose a working model for RPE65-membrane binding, and to provide a novel role for cysteine palmitoylation.

Core Temperature Lability Predicts Sympathetic Interruption and Cognitive Performance during Heat Exposure in Persons with Spinal Cord Injuries.

Among persons with high spinal cord injury (Hi-SCI: > T5), changes in core body temperature (Tcore) and cognitive performance during heat exposure appear related to degree of sympathetic interruption. Twenty men with Hi-SCI (C4-T4, American Spinal Injury Association Impairment Scale [AIS] A-B) and 19 matched, able-bodied controls were acclimated to 27°C baseline (BL) before exposure to 35°C heat challenge (HC). Two groups, differentiated by increase in Tcore during HC, were identified: high responders (HR-SCI: ΔTcore ≥0.5°C; n = 13, C4-T2) and low responders (LR-SCI: ΔTcore <0.5°C; n = 7, C4-T4). Tcore, distal skin temperatures (Tskavg), and distal microvascular perfusion (LDFboth feet) were measured, as were indices of sympathetic integrity, mean arterial pressure (MAP), and extremity sweat rate (SRavg). Cognitive performance was assessed at BL and post-HC, using the Stroop Color and Word and Wechsler Adult Intelligence Scale-Fourth Edition (WAIS-IV) Digit Span tests. At BL, Tcore of the HR-SCI group (36.6 ± 0.4°C) was lower than that for the LR-SCI (37.1 ± 0.3°C; p = 0.011) and control groups (37.3 ± 0.3°C; p < 0.001). After HC, Tcore was not different among groups. MAP of the HR-SCI group (70.9 ± 9.8 mm Hg) was lower than that of the LR-SCI (81.8 ± 7.0 mm Hg; p = 0.048) and control groups (89.9 ± 9.9 mm Hg; p < 0.001). SRavg increased more in the control group (77.0 ± 52.5 nL/cm2/min) than in the HR-SCI group (15.5 ± 22.0 nL/cm2/min; p = 0.001). Only the HR-SCI group had significant increases in T-Scores of Stroop Word (7.5 ± 4.4; p < 0.001), WAIS-IV Digit Span Sequence (1.9 ± 1.8; p = 0.002), and WAIS-IV Digit Span Total (1.4 ± 1.6; p = 0.008). Persons with SCI who responded to HC with a greater change in Tcore demonstrated evidence of greater sympathetic interruption and had an associated improvement in cognitive performance.

Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env.

As the sole viral antigen on the HIV-1-virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1-Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C 433C (DS) variant specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like-particle and soluble formats providing a new generation of vaccine antigens.