Connected to the pump

Mitochondrial complex I is a near–1 MDa membrane complex essential for energy metabolism. It is the first enzyme in the mitochondrial respiratory chain; it extracts energy from NADH and transforms it into a voltage across the mitochondrial membrane by use of its proton pump. Dysregulation can lead to the production of reactive oxygen species and neurodegenerative diseases. The mechanism for converting redox energy to proton pumping is not known in detail, partly due to a lack of a high-resolution structure of the whole complex. Previous electron microscopy studies revealed an L-shaped overall architecture, with a membrane arm and a peripheral arm, and the crystal structure of the peripheral arm from bacterial complex I suggested that all redox cofactors were localized in this arm. Now Hunte et al. present the X-ray crystallographic structure of mitochondrial complex I from the aerobic yeast Yarrowia lipolytica at a resolution of 6.3 Å. The structure confirms the L shape and shows an angle of about 100° between the arms. The peripheral arm contains the eight iron-sulfur clusters, with seven of them in a row forming a 'wire' linking catalytic sites. In the membrane arm, 71 a-helical transmembrane segments are present. At least two of the subunits, including one at the furthest point away from the peripheral arm, have structural similarities to the sodium-proton antiporter and therefore are likely components of the proton pump. The membrane arm is formed by two domains, proximal and distal, and linking these two is a 60-Å α-helix that lies parallel to the membrane arm. The distance between the redox site and the putative proton pump is too far for a direct mechanism to be probable. Instead, this long helix is likely to act as a key element transducing conformational energy to the proton pump. (Science, published online 1 July 2010, doi: 10.1126/science.1191046) MH

Smoothening Hh signaling with lipid

Hedgehog (Hh) signaling is critical for developmental events in multiple tissues and species, and defects in this pathway have been linked to disease, including cancer. Hh is unusual in that its receptor, Patched (Ptc), is actually a negative regulator of signaling downstream of Smoothened (Smo), which resembles a seven-pass transmembrane protein similar to G protein–coupled receptors. Upon Hh binding, Ptc repression of Smo is relieved, and signaling is thus activated. How Ptc inhibits Smo is currently unclear, and indeed, whether they physically interact remains an open question. However, it is known that membrane localization of Smo is regulated and appears to be affected by Ptc. Banerjee and colleagues have now genetically linked Drosophila melanogaster Smo activation status to levels of the phospholipid phosphatidylinositol-4 phosphate (PI4P). The authors used mutant analysis in developing eye and wing tissue to reduce Sac1 function (a homolog of the yeast phosphatase that reduces levels of cytoplasmic PI4P) and to show that Drosophila sac1 mutations result in increased PI4P immunostaining. sac1 mutant clones have higher levels of the Hh pathway transcriptional effector Ci as well as increased expression of transcriptional Hh targets. In addition, similar to an effect seen upon Hh signaling, sac1 mutant cells show increased Smo membrane localization. Indeed, reducing sac1 function has many features of active Hh signaling, and conversely, targeting the homolog of STT4, the yeast kinase that opposes sac1 function in PI4P metabolism, has hallmarks of loss of Hh signaling in Drosophila. In addition, Ptc mutants also affect PI4P levels, thus linking regulation of Smo through PI4P regulation to Ptc. Finally, the authors showed that targeting human STT4 homologs, but not sac1, results in altered Hh signaling in a mammalian reporter cell line, arguing that at least some aspects of this regulation might be conserved. Although the molecular mechanisms involved in PI4P regulation of Hh signaling are currently unclear, this work provides a new avenue along which to pursue Ptc inhibition of Smo, currently a black box in the regulation of Hh signaling and a point in this pathway that is affected by many human disease-linked mutations that have been related to Hh signaling. (Dev. Cell. 19, 54–65, 2010) SL

Neutralizing HIV-1

Highly active antiretroviral therapy (HAART) has been able to dramatically increase both the quality and length of life of an HIV-infected individual; however, progress toward an anti-HIV vaccine has been much slower than anticipated. In a pair of recent papers, a multicenter team of researchers identified and characterized a broadly neutralizing monoclonal antibody in work that represents an important step toward the development of effective anti-HIV vaccine. In the first paper, Wu et al. designed and generated several recombinant glycoproteins that functionally mimicked the CD4-binding site of the viral envelope component gp120. They then screened sera from HIV-positive patients and found reactive antibodies for the recombinant glycoproteins. Three antibodies isolated from individual B cells (VRC01, VRC02 and VRC03) bound gp120 with high affinity at the CD4 binding site. When screened against a panel of viruses pseudotyped with envelope from 190 different HIV-1 strains, both VRC01 and VRC02 neutralized ∼90% of the strains. In the second paper, Zhou et al. solved the X-ray crystal structure of the antigen-binding fragment of VRC01 bound to an HIV-1 gp120 core. As expected, VRC01 mimics many interactions seen in a previous crystal structure of the CD4–gp120 complex. However, a larger proportion of the interactions between VRC01 and gp120 occurs at the conformationally invariant outer domain of gp120, which may explain why VRC01 is so potent and broadly neutralizing. It is not clear whether VRC01 itself will be useful in the clinic, but it is hoped that the lessons learned from this work will facilitate the design of future anti-HIV vaccine candidates. (Science, published online 8 July 2010, doi: 10.1126/science.1187659 and doi: 10.1126/science.1192819) JMF

Written by Joshua M Finkelstein, Maria Hodges & Sabbi Lall