Saurav Misra

Mechanism of ubiquitin recognition by the CUE domain of Vps9p.

Coupling of ubiquitin conjugation to ER degradation (CUE) domains are approximately 50 amino acid monoubiquitin binding motifs found in proteins of trafficking and ubiquitination pathways. The 2.3 A structure of the Vps9p-CUE domain is a dimeric domain-swapped variant of the ubiquitin binding UBA domain. The 1.7 A structure of the CUE:ubiquitin complex shows that one CUE dimer binds one ubiquitin molecule. The bound CUE dimer is kinked relative to the unbound CUE dimer and wraps around ubiquitin. The CUE monomer contains two ubiquitin binding surfaces on opposite faces of the molecule that cannot bind simultaneously to a single ubiquitin molecule. Dimerization of the CUE domain allows both surfaces to contact a single ubiquitin molecule, providing a mechanism for high-affinity binding to monoubiquitin.

Structural basis for acidic-cluster-dileucine sorting-signal recognition by VHS domains

Specific sorting signals direct transmembrane proteins to the compartments of the endosomal–lysosomal system. Acidic-cluster-dileucine signals present within the cytoplasmic tails of sorting receptors, such as the cation-independent and cation-dependent mannose-6-phosphate receptors, are recognized by the GGA (Golgi-localized, γ-ear-containing, ADP-ribosylation-factor-binding) proteins. The VHS (Vps27p, Hrs and STAM) domains of the GGA proteins are responsible for the highly specific recognition of these acidic-cluster-dileucine signals. Here we report the structures of the VHS domain of human GGA3 complexed with signals from both mannose-6-phosphate receptors. The signals bind in an extended conformation to helices 6 and 8 of the VHS domain. The structures highlight an Asp residue separated by two residues from a dileucine sequence as critical recognition elements. The side chains of the Asp-X-X-Leu-Leu sequence interact with subsites consisting of one electropositive and two shallow hydrophobic pockets, respectively. The rigid spatial alignment of the three binding subsites leads to high specificity.

Recognizing Phosphatidylinositol 3-Phosphate

Phosphatidylinositol 3-phosphate directs the endosomal localization of regulatory proteins by binding to FYVE and PX domains. New structures of these domains complexed with the phosphoinositide headgroup show how interactions with phosphate and hydroxyl groups differentiate this lipid from all others.

Arginine-82 regulates the pKa of the group responsible for the light-driven proton release in bacteriorhodopsin.

In wild-type bacteriorhodopsin light-induced proton release occurs before uptake at neutral pH. In contrast, in mutants in which R82 is replaced by a neutral residue (as in R82A and R82Q), only a small fraction of the protons is released before proton uptake at neutral pH; the major fraction is released after uptake. In R82Q the relative amounts of the two types of proton release, early (preceding proton uptake) and late (following proton uptake), are pH dependent. The main conclusions are that 1) R82 is not the normal light-driven proton release group; early proton release can be observed in the R82Q mutant at higher pH values, suggesting that the proton release group has not been eliminated. 2) R82 affects the pKa of the proton release group both in the unphotolyzed state of the pigment and during the photocycle. In the wild type (in 150 mM salt) the pKa of this group decreases from approximately 9.5 in the unphotolyzed pigment to approximately 5.8 in the M intermediate, leading to early proton release at neutral pH. In the R82 mutants the respective values of pKa of the proton release group in the unphotolyzed pigment and in M are approximately 8 and 7.5 in R82Q (in 1 M salt) and approximately 8 and 6.5 in R82K (in 150 mM KCl). Thus in R82Q the pKa of the proton release group does not decrease enough in the photocycle to allow early proton release from this group at neutral pH. 3) Early proton release in R82Q can be detected as a photocurrent signal that is kinetically distinct from those photocurrents that are due to proton movements from the Schiff base to D85 during M formation and from D96 to the Schiff base during the M-->N transition. 4) In R82Q, at neutral pH, proton uptake from the medium occurs during the formation of O. The proton is released during the O-->bacteriorhodopsin transition, probably from D85 because the normal proton release group cannot deprotonate at this pH. 5) The time constant of early proton release is increased from 85 microseconds in the wild type to 1 ms in R82Q (in 150 mM salt). This can be directly attributed to the increase in the pKa of the proton release group and also explains the uncoupling of proton release from M formation. 6) In the E204Q mutant only late proton release is observed at both neutral and alkaline pH, consistent with the idea that E204 is the proton release group. The proton release is concurrent with the O-->bacteriorhodopsin transition, as in R82Q at neutral pH.

pH dependence of light-induced proton release by bacteriorhodopsin

We have measured the current generated by light‐activated proton release from bacteriorhodopsin into solution as a function of both pH and ionic strength. We find that proton release into solution decreases with increasing pH with an intrinsic pK a of 8.2 ± 0.2. This pH dependence indicates that the deprotonation of a certain group inhibits or abolishes proton release. Under physiological conditions, this group either releases a proton directly into solution or interacts with the site of proton release. The most immediate candidates for this protonatable species are tyrosine‐57, tyrosine‐185, arginine‐82, and water; acting individually or cooperatively. The salt dependence of the apparent pK a of this group also allows us to calculate the surface charge density of about −5 charges per bacteriorhodopsin, compatible with previous estimates.

Mutation of a Surface Residue, Lysine-129, Reverses the Order of Proton Release and Uptake in Bacteriorhodopsin; Guanidine Hydrochloride Restores It

K129 is a residue located in the extracellular loop connecting transmembrane helices D and E of bacteriorhodopsin. Replacement of K129 with a histidine alters the pKas of two key residues in the proton transport pathway, D85, and the proton release group (probably E204); the resulting pigment has properties that differ markedly from the wild type. 1) In the unphotolyzed state of the K129H mutant, the pKa of D85 is 5.1 +/- 0.1 in 150 mM KCl (compared to approximately 2.6 in the wild-type bacteriorhodopsin), whereas the unphotolyzed-state pKa of E204 decreases to 8.1 +/- 0.1 (from approximately 9.5 in the wild-type pigment). 2) The pKa of E204 in the M state is 7.0 +/- 0.1 in K129H, compared to approximately 5.8 in the wild-type pigment. 3) As a result of the change in the pKa of E204 in M, the order of light-induced proton release and uptake exhibits a dependence on pH in K129H differing from that of the wild type: at neutral pH and moderate salt concentrations (150 mM KCl), light-induced proton uptake precedes proton release, whereas it follows proton release at higher pH. This pumping behavior is similar to that seen in a related bacterial rhodopsin, archaerhodopsin-1, which has a histidine in the position analogous to K129. 4) At alkaline pH, a substantial fraction of all-trans K129H pigment (approximately 30%) undergoes a conversion into a shorter wavelength species, P480, with pKa approximately 8.1, close to the pKa of E204. 5) Guanidine hydrochloride lowers the pKas of D85 and E204 in the ground state and the pKa of E204 in the M intermediate, and restores the normal order of proton release before uptake at neutral pH. 6) In the K129H mutant the coupling between D85 and E204 is weaker than in wild-type bacteriorhodopsin. In the unphotolyzed pigment, the change in the pKas of either residue when the other changes its protonation state is only 1.5 units compared to 4.9 units in wild-type bacteriorhodopsin. In the M state of photolyzed K129H pigment, the corresponding change is 1 unit, compared to 3.7 units in the wild-type pigment. We suggest that K129 may be involved in stabilizing the hydrogen bonding network that couples E204 and D85. Substitution of K129 with a histidine residue causes structural changes that alter this coupling and affect the pKas of E204 and D85.

Structural genomics and signaling domains

Many novel signal transduction domains are being identified in the wake of genome sequencing projects and improved sensitivity in homology-detection techniques. The functions of these domains are being discovered by hypothesis-driven experiments and structural genomics approaches. This article reviews the recent highlights of research on modular signaling domains, and the relative contributions and limitations of the various approaches being used.

Contribution of Proton Release to the B2 Photocurrent of Bacteriorhodopsin

The contribution of proton release from the so-called proton release group to the microsecond B2 photocurrent from bacteriorhodopsin (bR) oriented in polyacrylamide gels was determined. The fraction of the B2 current due to proton release was resolved by titration of the proton release group in M. At pH values below the pKa of the proton release group in M, the proton release group cannot release its proton during the first half of the bacteriorhodopsin photocycle. At these pH values, the B2 photocurrent is due primarily to translocation of the Schiff base proton to Asp85. The B2 photocurrent was measured in wild-type bR gels at pH 4.5-7.5, in 100 mM KCl/50 mM phosphate. The B2 photocurrent area (proportional to the amount of charge moved) exhibits a pH dependence with a pKa of 6.1. This is suggested to be the pKa of the proton release group in M; the value obtained is in good agreement with previous results obtained by examining photocycle kinetics and pH-sensitive dye signals. In the mutant Glu204Gln, the B2 photocurrent of the mutant membranes was pH independent between pH 4 and 7. Because the proton release group is incapacitated, and early proton release is eliminated in the Glu204Gln mutant, this supports the idea that the pH dependence of the B2 photocurrent in the wild type reflects the titration of the proton release group. In wild-type bacteriorhodopsin, proton release contributes approximately half of the B2 area at pH 7.5. The B2 area in the Glu204Gln mutant is similar to that in the wild type at pH 4.5; in both cases, the B2 current is likely due only to movement of the Schiff base proton to Asp85.

Mutation of Arginine 134 to Lysine Alters the pKas of Key Groups Involved in Proton Pumping by Bacteriorhodopsin

Abstract— Arginine 134 is located near the extracellular surface of bacteriorhodopsin (bR) and may interact with one or more nearby glutamate residues. In the bR mutant R134K, light‐induced Schiff‐base deprotonation (formation of the M intermediate) exhibits several kinetic components and has a complex pH dependence. The kinetics and pH dependence of M formation were analyzed using the following general guidelines for interpreting M formation: (1) The fastest component of M formation reflects the redistribution of the Schiff‐base proton to D85, the usual proton acceptor, in response to the change in the proton affinities of the Schiff base and D85 early in the photocycle; (2) Two additional components of M formation reflect transitions between spectroscopically similar substates of M. By applying these guidelines, supplemented by information about the pKas of D85 and the proton release group from acid (purple‐to‐blue) and alkaline titrations of the absorption spectra of the unphotolyzed R134K pigment, we explain the pH dependence of M formation as being due to titration of the counter‐ion, D85, and of the proton release group. We calculate, in R134K, that the pKa of D85 is 4.6 in the unphotolyzed state, while the pKa of the proton release group is 8.0 in the unphotolyzed state but drops to ?5.8 in the M intermediate. The same value for the pKa of the proton release group in the M intermediate is obtained when we use photocurrent measurements to monitor proton release.

Charge Movements in the 13-cis Photocycles of the Bacteriorhodopsin Mutants R82K and R82Q

We have examined light‐induced currents in oriented membranes of the bacteriorhodopsin mutants R82K and R82Q. Our results suggest that two photocurrent components found in R82K, with 30 and 300 us lifetimes, are due to the photocycle of the 13‐cis rather than the all‐trans form of the pigment. We investigated the pH dependence of these components and their correspondence to absorbance changes at 660 nm characteristic of pho‐tointermediates of the 13‐cis cycle. The presence of a D2O effect suggests that the charge motions producing these photocurrents are related to proton or protonated amino acid movement within the molecule. The current amplitudes depend on the protonation states of at least two residues, D85 and (probably) E204. In R82Q, a 10 pis photocurrent is observed that also depends on the protonation state of D85 and is similar to the 30 us current in R82K. We attempt to explain these currents in terms of a model for interacting residues in the extracellular half of the bacteriorhodopsin channel.