Larry J. W. Miercke

Crystal structure of human aquaporin 4 at 1.8 A and its mechanism of conductance.

Aquaporin (AQP) 4 is the predominant water channel in the mammalian brain, abundantly expressed in the blood–brain and brain–cerebrospinal fluid interfaces of glial cells. Its function in cerebral water balance has implications in neuropathological disorders, including brain edema, stroke, and head injuries. The 1.8-Å crystal structure reveals the molecular basis for the water selectivity of the channel. Unlike the case in the structures of water-selective AQPs AqpZ and AQP1, the asparagines of the 2 Asn-Pro-Ala motifs do not hydrogen bond to the same water molecule; instead, they bond to 2 different water molecules in the center of the channel. Molecular dynamics simulations were performed to ask how this observation bears on the proposed mechanisms for how AQPs remain totally insulating to any proton conductance while maintaining a single file of hydrogen bonded water molecules throughout the channel.

Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: Versatility from a unique substrate channel

As the first structural elucidation of a modular polyketide synthase (PKS) domain, the crystal structure of the macrocycle-forming thioesterase (TE) domain from the 6-deoxyerythronolide B synthase (DEBS) was solved by a combination of multiple isomorphous replacement and multiwavelength anomalous dispersion and refined to an R factor of 24.1% to 2.8-Å resolution. Its overall tertiary architecture belongs to the α/β-hydrolase family, with two unusual features unprecedented in this family: a hydrophobic leucine-rich dimer interface and a substrate channel that passes through the entire protein. The active site triad, comprised of Asp-169, His-259, and Ser-142, is located in the middle of the substrate channel, suggesting the passage of the substrate through the protein. Modeling indicates that the active site can accommodate and orient the 6-deoxyerythronolide B precursor uniquely, while at the same time shielding the active site from external water and catalyzing cyclization by macrolactone formation. The geometry and organization of functional groups explain the observed substrate specificity of this TE and offer strategies for engineering macrocycle biosynthesis. Docking of a homology model of the upstream acyl carrier protein (ACP6) against the TE suggests that the 2-fold axis of the TE dimer may also be the axis of symmetry that determines the arrangement of domains in the entire DEBS. Sequence conservation suggests that all TEs from modular polyketide synthases have a similar fold, dimer 2-fold axis, and substrate channel geometry.

Crystal structure of the aquaglyceroporin PfAQP from the malarial parasite Plasmodium falciparum.

The 2.05-Å resolution structure of the aquaglyceroporin from the malarial parasite Plasmodium falciparum (PfAQP), a protein important in the parasites life cycle, has been solved. The structure provides key evidence for the basis of water versus glycerol selectivity in aquaporin family members. Unlike its closest homolog of known structure, GlpF, the channel conducts both glycerol and water at high rates, framing the question of what determines high water conductance in aquaporin channels. The universally conserved arginine in the selectivity filter is constrained by only two hydrogen bonds in GlpF, whereas there are three in all water-selective aquaporins and in PfAQP. The decreased cost of dehydrating the triply-satisfied arginine cation may provide the basis for high water conductance. The two Asn-Pro-Ala (NPA) regions of PfAQP, which bear rare substitutions to Asn-Leu-Ala (NLA) and Asn-Pro-Ser (NPS), participate in preserving the orientation of the selectivity filter asparagines in the center of the channel.

Catalysis, Specificity, and ACP Docking Site of Streptomyces coelicolor Malonyl-CoA:ACP Transacylase

Malonyl-CoA:ACP transacylase (MAT), the fabD gene product of Streptomyces coelicolor A3(2), participates in both fatty acid and polyketide synthesis pathways, transferring malonyl groups that are used as extender units in chain growth from malonyl-CoA to pathway-specific acyl carrier proteins (ACPs). Here, the 2.0 A structure reveals an invariant arginine bound to an acetate that mimics the malonyl carboxylate and helps define the extender unit binding site. Catalysis may only occur when the oxyanion hole is formed through substrate binding, preventing hydrolysis of the acyl-enzyme intermediate. Macromolecular docking simulations with actinorhodin ACP suggest that the majority of the ACP docking surface is formed by a helical flap. These results should help to engineer polyketide synthases (PKSs) that produce novel polyketides.

A general protocol for the crystallization of membrane proteins for X-ray structural investigation

Protein crystallography is used to generate atomic resolution structures of protein molecules. These structures provide information about biological function, mechanism and interaction of a protein with substrates or effectors including DNA, RNA, cofactors or other small molecules, ions and other proteins. This technique can be applied to membrane proteins resident in the membranes of cells. To accomplish this, membrane proteins first need to be either heterologously expressed or purified from a native source. The protein has to be extracted from the lipid membrane with a mild detergent and purified to a stable, homogeneous population that may then be crystallized. Protein crystals are then used for X-ray diffraction to yield atomic resolution structures of the desired membrane protein target. Below, we present a general protocol for the growth of diffraction quality membrane protein crystals. The process of protein crystallization is highly variable, and obtaining diffraction quality crystals can require weeks to months or even years in some cases.

Tandem Facial Amphiphiles for Membrane Protein Stabilization

We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles.

Crystal Structure of the Priming β-Ketosynthase from the R1128 Polyketide Biosynthetic Pathway

ZhuH is a priming ketosynthase that initiates the elongation of the polyketide chain in the biosynthetic pathway of a type II polyketide, R1128. The crystal structure of ZhuH in complex with the priming substrate acetyl-CoA reveals an extensive loop region at the dimer interface that appears to affect the selectivity for the primer unit. Acetyl-CoA is bound in a 20 A-long channel, which placed the acetyl group against the catalytic triad. Analysis of the primer unit binding site in ZhuH suggests that it can accommodate acyl chains that are two to four carbons long. Selectivity and primer unit size appear to involve the side chains of three residues on the loops close to the dimer interface that constitute the bottom of the substrate binding pocket.

Glycerol facilitator GlpF and the associated aquaporin family of channels

The aqua (glycero) porins conduct water (and glycerol) across cell membranes. The structure of these channels reveals a tripathic channel that supports a hydrophobic surface and, opposite to this, a line of eight hydrogen-bond acceptors and four hydrogen-bond donors. The eight carbonyls act as acceptors for water (or glycerol OH) molecules. The central water molecule in the channel is oriented to polarize hydrogen atoms outward from the center. This arrangement suggests how the structure prevents the potentially lethal conduction of protons across the membrane. The structure also suggests the mechanism behind the selectivity of aquaglyceroporins for glycerol, the basis for enantioselectivity among alditols, and the basis for the prevention of any leakage of the electrochemical gradient.

Structural context shapes the aquaporin selectivity filter

Aquaporins are transmembrane channels that facilitate the permeation of water and small, uncharged amphipathic molecules across cellular membranes. One distinct aquaporin subfamily contains pure water channels, whereas a second subfamily contains channels that conduct small alditols such as glycerol, in addition to water. Distinction between these substrates is central to aquaporin function, though the contributions of protein structural motifs required for selectivity are not yet fully characterized. To address this question, we sequentially engineered three signature amino acids of the glycerol-conducting subfamily into the Escherichia coli water channel aquaporin Z (AqpZ). Functional analysis of these mutant channels showed a decrease in water permeability but not the expected increase in glycerol conduction. Using X-ray crystallography, we determined the atomic resolution structures of the mutant channels. The structures revealed a channel surprisingly similar in size to the wild-type AqpZ pore. Comparison with measured rates of transport showed that, as the size of the selectivity filter region of the channel approaches that of water, channel hydrophilicity dominated water conduction energetics. In contrast, the major determinant of selectivity for larger amphipathic molecules such as glycerol was channel cross-section size. Finally, we find that, although the selectivity filter region is indeed central to substrate transport, other structural elements that do not directly interact with the substrates, such as the loop connecting helices M6 and M7, and the C loop between helices C4 and C5, play an essential role in facilitating selectivity.

Selectivity and conductance among the glycerol and water conducting aquaporin family of channels

The atomic structures of a transmembrane water plus glycerol conducting channel (GlpF), and now of aquaporin Z (AqpZ) from the same species, Escherichia coli, bring the total to three atomic resolution structures in the aquaporin (AQP) family. Members of the AQP family each assemble as tetramers of four channels. Common helical axes support a wider channel in the glycerol plus water channel paradigm, GlpF. Water molecules form a single hydrogen bonded file throughout the 28 Å long channel in AqpZ. The basis for absolute exclusion of proton or hydronium ion conductance through the line of water is explored using simulations.