Structure (v.13, #8)

Allostery Trumps Antibiotic Resistance by Gerard D. Wright (1089-1090).
In this issue of Structure, Kohl and colleagues report on the structure of an antibiotic resistance kinase in complex with an inhibitor selected from an ankyrin repeat library that binds the enzyme with high affinity and reverses antibiotic resistance in vivo ().

A GLYmmer of Insight into Fibril Formation by Vladimir N. Uversky (1090-1092).
Identifying factors contributing to protein aggregation and amyloid fibril formation is essential for understanding protein deposition diseases. In this issue of Structure, Chiti and colleagues () use acylphosphatase as a model to describe a new, protective role for glycine.

Bending and Cutting Forks and Flaps by Gregory D. Van Duyne (1092-1093).
Recent advances in the structural biology and biochemistry of two archaeal DNA repair nucleases (, in this issue of Structure) have shed new light on how these systems achieve extraordinary specificity for branched DNA substrates.

Bend to Open? by Daniel L. Minor (1094-1095).
In this issue of Structure, report the 9.6 Å structure of the ryanodine receptor (RyR1) closed state. The structure shows a bent inner helix and raises the question of whether inner helix deformation is really a conserved channel gating mechanism.

The homologous bacterially expressed cholesterol-dependent cytolysins (CDCs) form pores via oligomerization; this must occur preferentially once the target membrane has been engaged. Conformational changes in CDCs then drive partition from an aqueous environment to a lipidic one. This review addresses how premature oligomerization is prevented, how conformational changes are triggered, and how cooperativity between subunits brings about new functionality absent from isolated protomers. Variations are found in the answers provided by the CDCs to these issues. Some toxins use pH as a trigger of activity, but recent results have shown that dimerization in solution is an alternative way of preventing premature oligomerization, in particular for the CDC from Clostridium perfringens, perfringolysin. More controversially, there is still no resolution to the debate as to whether incomplete (arciform) oligomers form pores: recent results again suggest that they do.

What Makes an Aquaporin a Glycerol Channel? A Comparative Study of AqpZ and GlpF by Yi Wang; Klaus Schulten; Emad Tajkhorshid (1107-1118).
The recent availability of high-resolution structures of two structurally highly homologous, but functionally distinct aquaporins from the same species, namely Escherichia coli AqpZ, a pure water channel, and GlpF, a glycerol channel, presents a unique opportunity to understand the mechanism of substrate selectivity in these channels. Comparison of the free energy profile of glycerol conduction through AqpZ and GlpF reveals a much larger barrier in AqpZ (22.8 kcal/mol) than in GlpF (7.3 kcal/mol). In either channel, the highest barrier is located at the selectivity filter. Analysis of substrate-protein interactions suggests that steric restriction of AqpZ is the main contribution to this large barrier. Another important difference is the presence of a deep energy well at the periplasmic vestibule of GlpF, which was not found in AqpZ. The latter difference can be attributed to the more pronounced structural asymmetry of GlpF, which may play a role in attracting glycerol.

The Molecular Origins of Specificity in the Assembly of a Multienzyme Complex by René A.W. Frank; J. Venkatesh Pratap; Xue Y. Pei; Richard N. Perham; Ben F. Luisi (1119-1130).
The pyruvate dehydrogenase (PDH) multienzyme complex is central to oxidative metabolism. We present the first crystal structure of a complex between pyruvate decarboxylase (E1) and the peripheral subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2). The interface is dominated by a “charge zipper” of networked salt bridges. Remarkably, the PSBD uses essentially the same zipper to alternately recognize the dihydrolipoyl dehydrogenase (E3) component of the PDH assembly. The PSBD achieves this dual recognition largely through the addition of a network of interfacial water molecules unique to the E1-PSBD complex. These structural comparisons illuminate our observations that the formation of this water-rich E1-E2 interface is largely enthalpy driven, whereas that of the E3-PSBD complex (from which water is excluded) is entropy driven. Interfacial water molecules thus diversify surface complementarity and contribute to avidity, enthalpically. Additionally, the E1-PSBD structure provides insight into the organization and active site coupling within the ∼9 MDa PDH complex.

Allosteric Inhibition of Aminoglycoside Phosphotransferase by a Designed Ankyrin Repeat Protein by Andreas Kohl; Patrick Amstutz; Petra Parizek; H. Kaspar Binz; Christophe Briand; Guido Capitani; Patrik Forrer; Andreas Plückthun; Markus G. Grütter (1131-1141).
Aminoglycoside phosphotransferase (3′)-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. We report here the crystal structure of APH, trapped in an inactive conformation by a tailor-made inhibitory ankyrin repeat (AR) protein, at 2.15 Å resolution. The inhibitor was selected from a combinatorial library of designed AR proteins. The AR protein binds the C-terminal lobe of APH and thereby stabilizes three α helices, which are necessary for substrate binding, in a significantly displaced conformation. BIAcore analysis and kinetic enzyme inhibition experiments are consistent with the proposed allosteric inhibition mechanism. In contrast to most small-molecule kinase inhibitors, the AR proteins are not restricted to active site binding, allowing for higher specificity. Inactive conformations of pharmaceutically relevant enzymes, as can be elucidated with the approach presented here, represent powerful starting points for rational drug design.

Glycine Residues Appear to Be Evolutionarily Conserved for Their Ability to Inhibit Aggregation by Claudia Parrini; Niccolò Taddei; Matteo Ramazzotti; Donatella Degl’Innocenti; Giampietro Ramponi; Christopher M. Dobson; Fabrizio Chiti (1143-1151).
Six glycine residues of human muscle acylphosphatase (AcP) are evolutionarily conserved across the three domains of life. We have generated six variants of AcP, each having a glycine substituted by an alanine (G15A, G19A, G37A, G45A, G53A, and G69A). Three additional variants had Gly45 replaced by serine, glutamate, and arginine, respectively. The mutational variants do not, on average, have a lower conformational stability than other variants with substitutions of nonconserved residues. In addition, only the G15A variant is enzymatically inactive. However, all variants, with the exception of the G15A mutant, form amyloid aggregates more rapidly than the wild-type. Dynamic light-scattering experiments carried out under conditions close to physiological confirm that aggregate formation is generally more pronounced for the glycine-substituted variants. Apart from the glycine at position 15, all other conserved glycine residues in this protein could have been maintained during evolution because of their ability to inhibit aggregation.

Given any operational definition of pairwise interaction, the set of residues that differ between two structurally homologous proteins can be uniquely partitioned into subsets of clusters for which no such interactions occur between clusters. Although hybrid protein sequences that preserve such clustering are consistent with tertiary structures composed of only parental native-like interactions, the stability of such predicted structures will depend upon the physical robustness of the assumed interaction potential. A simple distance cutoff criterion was applied to the most thermostable protein known to predict such a seven-residue cluster in the metal binding site region of Pyrococcus furiosus rubredoxin and a mesophile homolog. Both conformational stability and thermal transition temperature measurements demonstrate that 39% of the differential stability arises from these seven residues.

Atomic Models by Cryo-EM and Site-Directed Spin Labeling: Application to the N-Terminal Region of Hsp16.5 by Hanane A. Koteiche; Steve Chiu; Rebecca L. Majdoch; Phoebe L. Stewart; Hassane S. Mchaourab (1165-1171).
We report an approach for determining the structure of macromolecular assemblies by the combined application of cryo-electron microscopy (cryo-EM) and site-directed spin labeling electron paramagnetic resonance spectroscopy (EPR). This approach is illustrated for Hsp16.5, a small heat shock protein that prevents the aggregation of nonnative proteins. The structure of Hsp16.5 has been previously studied by both cryo-EM and X-ray crystallography. The crystal structure revealed a roughly spherical protein shell with dodecameric symmetry; however, residues 1–32 were found to be disordered. The cryo-EM reconstruction at 13 Å resolution appeared similar to the crystal structure but with additional internal density corresponding to the N-terminal regions of the 24 subunits. In this study, a systematic application of site-directed spin labeling and EPR spectroscopy was carried out. By combining the EPR constraints from spin label accessibilities and proximities with the cryo-EM density, we obtained an atomic model for a portion of the Hsp16.5 N-terminal region in the context of the oligomeric complex.

Conferring Substrate Specificity to DNA Helicases: Role of the RecQ HRDC Domain by Douglas A. Bernstein; James L. Keck (1173-1182).
RecQ DNA helicases are multidomain enzymes that play pivotal roles in genome maintenance pathways. While the ATPase and helicase activities of these enzymes can be attributed to the conserved catalytic core domain, the role of the Helicase-and-RNase-D-C-terminal (HRDC) domain in RecQ function has yet to be elucidated. Here, we report the crystal structure of the E. coli RecQ HRDC domain, revealing a globular fold that resembles known DNA binding domains. We show that this domain preferentially binds single-stranded DNA and identify its DNA binding surface. HRDC domain mutations in full-length RecQ lead to surprising differences in its structure-specific DNA binding properties. These data support a model in which naturally occurring variations in DNA binding residues among diverse RecQ homologs serve to target these enzymes to distinct substrates and provide insight into a mechanism whereby RecQ enzymes have evolved distinct functions in organisms that encode multiple recQ genes.

Structural and Functional Analyses of an Archaeal XPF/Rad1/Mus81 Nuclease: Asymmetric DNA Binding and Cleavage Mechanisms by Tatsuya Nishino; Kayoko Komori; Yoshizumi Ishino; Kosuke Morikawa (1183-1192).
XPF/Rad1/Mus81/Hef proteins recognize and cleave branched DNA structures. XPF and Rad1 proteins cleave the 5′ side of nucleotide excision repair bubble, while Mus81 and Hef cleave similar sites of the nicked Holliday junction, fork, or flap structure. These proteins all function as dimers and consist of catalytic and helix-hairpin-helix DNA binding (HhH) domains. We have determined the crystal structure of the HhH domain of Pyrococcus furiosus Hef nuclease (HefHhH), which revealed the distinct mode of protein dimerization. Our structural and biochemical analyses also showed that each of the catalytic and HhH domains binds to distinct regions within the fork-structured DNA: each HhH domain from two separate subunits asymmetrically binds to the arm region, while the catalytic domain binds near the junction center. Upon binding to DNA, Hef nuclease disrupts base pairs near the cleavage site. It is most likely that this bipartite binding mode is conserved in the XPF/Rad1/Mus81 nuclease family.

NMR Structural Characterization and Computational Predictions of the Major Intermediate in Oxidative Folding of Leech Carboxypeptidase Inhibitor by Joan L. Arolas; Loyola D’Silva; Grzegorz M. Popowicz; Francesc X. Aviles; Tad A. Holak; Salvador Ventura (1193-1202).
The III-A intermediate constitutes the major rate-determining step in the oxidative folding of leech carboxypeptidase inhibitor (LCI). In this work, III-A has been directly purified from the folding reaction and structurally characterized by NMR spectroscopy. This species, containing three native disulfides, displays a highly native-like structure; however, it lacks some secondary structure elements, making it more flexible than native LCI. III-A represents a structurally determined example of a disulfide-insecure intermediate; direct oxidation of this species to the fully native protein seems to be restricted by the burial of its two free cysteine residues inside a native-like structure. We also show that theoretical approaches based on topological constraints predict with good accuracy the presence of this folding intermediate. Overall, the derived results suggest that, as it occurs with non-disulfide bonded proteins, native-like interactions between segments of secondary structure rather than the crosslinking of disulfide bonds direct the folding of LCI.

The Pore Structure of the Closed RyR1 Channel by Steven J. Ludtke; Irina I. Serysheva; Susan L. Hamilton; Wah Chiu (1203-1211).
Using single particle electron cryomicroscopy, several helices in the membrane-spanning region of RyR1, including an inner transmembrane helix, a short pore helix, and a helix parallel to the membrane on the cytoplasmic side, have been clearly resolved. Our model places a highly conserved glycine (G4934) at the hinge position of the bent inner helix and two rings of negative charges at the luminal and cytoplasmic mouths of the pore. The kinked inner helix closely resembles the inner helix of the open MthK channel, suggesting that kinking alone does not open RyR1, as proposed for K+ channels.

The quest to order and classify protein structures has lead to various classification schemes, focusing mostly on hierarchical relationships between structural domains. At the coarsest classification level, such schemes typically identify hundreds of types of fundamental units called folds. As a result, we picture protein structure space as a collection of isolated fold islands. It is obvious, however, that many protein folds share structural and functional commonalities. Locating those commonalities is important for our understanding of protein structure, function, and evolution. Here, we present an alternative view of the protein fold space, based on an interfold similarity measure that is related to the frequency of fragments shared between folds. In this view, protein structures form a complicated, crossconnected network with very interesting topology. We show that interfold similarity based on sequence/structure fragments correlates well with similarities of functions between protein populations in different folds.

Nucleotide-Induced DNA Polymerase Active Site Motions Accommodating a Mutagenic DNA Intermediate by Vinod K. Batra; William A. Beard; David D. Shock; Lars C. Pedersen; Samuel H. Wilson (1225-1233).
DNA polymerases occasionally insert the wrong nucleotide. For this error to become a mutation, the mispair must be extended. We report a structure of DNA polymerase β (pol β) with a DNA mismatch at the boundary of the polymerase active site. The structure of this complex indicates that the templating adenine of the mispair stacks with the primer terminus adenine while the templating (coding) cytosine is flipped out of the DNA helix. Soaking the crystals of the binary complex with dGTP resulted in crystals of a ternary substrate complex. In this case, the templating cytosine is observed within the DNA helix and forms Watson-Crick hydrogen bonds with the incoming dGTP. The adenine at the primer terminus has rotated into a syn-conformation to interact with the opposite adenine in a planar configuration. Yet, the 3′-hydroxyl on the primer terminus is out of position for efficient nucleotide insertion.