Functional variants in the LRRK2 gene confer shared effects on risk for Crohn's disease and Parkinson's disease

Crohn’s disease (CD), a form of inflammatory bowel disease, has a higher prevalence in Ashkenazi Jewish than in non-Jewish European populations. To define the role of nonsynonymous mutations, we performed exome sequencing of Ashkenazi Jewish patients with CD, followed by array-based genotyping and association analysis in 2066 CD cases and 3633 healthy controls. We detected association signals in the LRRK2 gene that conferred risk for CD (N2081D variant, P = 9.5 × 10−10) or protection from CD (N551K variant, tagging R1398H-associated haplotype, P = 3.3 × 10−8). These variants affected CD age of onset, disease location, LRRK2 activity, and autophagy. Bayesian network analysis of CD patient intestinal tissue further implicated LRRK2 in CD pathogenesis. Analysis of the extended LRRK2 locus in 24,570 CD cases, patients with Parkinson’s disease (PD), and healthy controls revealed extensive pleiotropy, with shared genetic effects between CD and PD in both Ashkenazi Jewish and non-Jewish cohorts. The LRRK2 N2081D CD risk allele is located in the same kinase domain as G2019S, a mutation that is the major genetic cause of familial and sporadic PD. Like the G2019S mutation, the N2081D variant was associated with increased kinase activity, whereas neither N551K nor R1398H variants on the protective haplotype altered kinase activity. We also confirmed that R1398H, but not N551K, increased guanosine triphosphate binding and hydrolyzing enzyme (GTPase) activity, thereby deactivating LRRK2. The presence of shared LRRK2 alleles in CD and PD provides refined insight into disease mechanisms and may have major implications for the treatment of these two seemingly unrelated diseases

Drug binding revealed by tandem mass spectrometry of a protein-micelle complex.

The protein-micelle complex formed between the protein EmrE and the lipid dodecylmaltoside has been examined by mass spectrometry. The results show that despite the unfavorable hydrophobic environment in the mass spectrometer it is possible to preserve protein submicelle complexes in the gas phase. The peaks assigned to the submicelle complexes are broad in nature and consistent with a heterogeneous distribution of lipid molecules attached to the protein complex. As such, the spectrum cannot be interpreted. To simplify this complexity we used a tandem mass spectrometry procedure in which discrete m/z values are isolated from the peak and subjected to collision-induced dissociation. These spectra reveal clusters of DDM molecules as well as sequential release of TPP+ and EmrE from the complex as the collision cell voltage is raised. Taken together, the results provide direct evidence for drug binding within a relevant gas-phase protein-micelle complex

The erythropoietin receptor transmembrane domain mediates complex formation with viral anemic and polycythemic gp55 proteins

Erythropoietin receptor (EpoR) activation is crucial for mature red blood cell production. The murine EpoR can also be activated by the envelope protein of the polycythemic (P) spleen focus forming virus (SFFV), gp55-P. Due to differences in the TM sequence, gp55 of the anemic (A) strain SFFV, gp55-A, cannot efficiently activate the EpoR. Using antibody-mediated immunofluorescence co-patching, we show that the majority of EpoR forms hetero-oligomers at the cell surface with gp55-P and, surprisingly, with gp55-A. The EpoR TM domain is targeted by gp55-P and -A, as only chimeric receptors containing EpoR TM sequences oligomerized with gp55 proteins. Both gp55-P and gp55-A are homodimers on the cell surface, as shown by co-patching. However, when the homomeric interactions of the isolated TM domains were assayed by TOXCAT bacterial reporter system, only the TM sequence of gp55-P was dimerized. Thus, homo-oligomerization of gp55 proteins is insufficient for full EpoR activation, and a correct conformation of the dimer in the TM region is required. This is supported by the failure of gp55-A3P, a mutant protein whose TM domain can homo-oligomerize, to fully activate EpoR. As unliganded EpoR forms TM-dependent but inactive homodimers, we propose that the EpoR can be activated to different extents by homodimeric gp55 proteins, depending on the conformation of the gp55 protein dimer in the TM region

The Cα—H⋅⋅⋅O hydrogen bond: A determinant of stability and specificity in transmembrane helix interactions

The Cα—H⋅⋅⋅O hydrogen bond has been given little attention as a determinant of transmembrane helix association. Stimulated by recent calculations suggesting that such bonds can be much stronger than has been supposed, we have analyzed 11 known membrane protein structures and found that apparent carbon α hydrogen bonds cluster frequently at glycine-, serine-, and threonine-rich packing interfaces between transmembrane helices. Parallel right-handed helix–helix interactions appear to favor Cα—H⋅⋅⋅O bond formation. In particular, Cα—H⋅⋅⋅O interactions are frequent between helices having the structural motif of the glycophorin A dimer and the GxxxG pair. We suggest that Cα—H⋅⋅⋅O hydrogen bonds are important determinants of stability and, depending on packing, specificity in membrane protein folding

Structural evidence for dimerization-regulated activation of an integral membrane phospholipase

Dimerization is a biological regulatory mechanism employed by both soluble and membrane proteins. However, there are few structural data on the factors that govern dimerization of membrane proteins. Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme which participates in secretion of colicins in Escherichia coli. In Campilobacter and Helicobacter pylori strains, OMPLA is implied in virulence. Its activity is regulated by reversible dimerization. Here we report X-ray structures of monomeric and dimeric OMPLA from E. coli. Dimer interactions occur almost exclusively in the apolar membrane-embedded parts, with two hydrogen bonds within the hydrophobic membrane area being key interactions. Dimerization results in functional oxyanion holes and substrate-binding pockets, which are absent in monomeric OMPLA. These results provide a detailed view of activation by dimerization of a membrane protein.

Modulation of Substrate Efflux in Bacterial Small Multidrug Resistance Proteins by Mutations at the Dimer Interface ▿

Bacteria evade the effects of cytotoxic compounds through the efflux activity of membrane-bound transporters such as the small multidrug resistance (SMR) proteins. Consisting typically of ca. 110 residues with four transmembrane (TM) α-helices, crystallographic studies have shown that TM helix 1 (TM1) through TM helix 3 (TM3) of each monomer create a substrate binding “pocket” within the membrane bilayer, while a TM4-TM4 interaction accounts for the primary dimer formation. Previous work from our lab has characterized a highly conserved small-residue heptad motif in the Halobacterium salinarum transporter Hsmr as 90GLXLIXXGV98 that lies along the TM4-TM4 dimer interface of SMR proteins as required for function. Focusing on conserved positions 91, 93, 94, and 98, we substituted the naturally occurring Hsmr residue for Ala, Phe, Ile, Leu, Met, and Val at each position in the Hsmr TM4-TM4 interface. Large-residue replacements were studied for their ability to dimerize on SDS-polyacrylamide gels, to bind the cytotoxic compound ethidium bromide, and to confer resistance by efflux. Although the relative activity of mutants did not correlate with dimer strength for all mutants, all functional mutants lay within 10% of dimerization relative to the wild type (WT), suggesting that the optimal dimer strength at TM4 is required for proper efflux. Furthermore, nonfunctional substitutions at the center of the dimerization interface that do not alter dimer strength suggest a dynamic TM4-TM4 “pivot point” that responds to the efflux requirements of different substrates. This functionally critical region represents a potential target for inhibiting the ability of bacteria to evade the effects of cytotoxic compounds