Chemical synthesis and enzymatic properties of RNase A analogues designed to enhance second-step catalytic activity.

In this paper, we have used total chemical synthesis of RNase A analogues in order to probe the molecular basis of enzyme catalysis. Our goal was to obligately fill the adenine-binding pocket on the enzyme molecule, and to thus pre-orient the imidazole side chain of His119 in its catalytically productive orientation. Two designed analogues of the RNase A protein molecule that contained an adenine moiety covalently bound to distinct amino acid side chains adjacent to the adenine binding pocket were prepared. A crystal structure of one analogue was determined at 2.3 Å resolution. Kinetic data for RNA transphosporylation and 2',3' cyclic mononucleotide hydrolysis were acquired for the adenine-containing RNase A analogue proteins. As anticipated, the presence of a covalently attached adenine on the enzyme molecule decreased the rate of transphosphorylation and increased the rate of hydrolysis, although the magnitude of the effects was small. This work illustrates the use of total protein synthesis to investigate the chemistry of enzyme catalysis in ways not possible through traditional biochemistry or molecular biology.

Role of a salt bridge in the model protein crambin explored by chemical protein synthesis: X-ray structure of a unique protein analogue, [V15A]crambin-alpha-carboxamide.

We have used total chemical synthesis to prepare [V15A]crambin-alpha-carboxamide, a unique protein analogue that eliminates a salt bridge between the delta-guanidinium of the Arg(10) side chain and the alpha-carboxylate of Asn(46) at the C-terminus of the polypeptide chain. This salt bridge is thought to be important for the folding and stability of the crambin protein molecule. Folding, with concomitant disulfide bond formation, of the fully reduced [V15A]crambin-alpha-carboxamide polypeptide was less efficient than folding/disulfide formation for the [V15A]crambin polypeptide under a standard set of conditions. To probe the origin of this less efficient folding/disulfide bond formation, we separately crystallized purified synthetic [V15A]crambin-alpha-carboxamide and chemically synthesized [V15A]crambin and solved their X-ray structures. The crystal structure of [V15A]crambin-alpha-carboxamide showed that elimination of the Arg(10)-Asn(46) salt bridge caused disorder of the C-terminal region of the polypeptide chain and affected the overall 'tightness' of the structure of the protein molecule. These studies, enabled by chemical protein synthesis, strongly suggest that in native crambin the Arg(10)-Asn(46) salt bridge contributes to efficient formation of correct disulfide bonds and also to the well-ordered structure of the protein molecule.

Structural and spectropotentiometric analysis of Blastochloris viridis heterodimer mutant reaction center.

Heterodimer mutant reaction centers (RCs) of Blastochloris viridis were crystallized using microfluidic technology. In this mutant, a leucine residue replaced the histidine residue which had acted as a fifth ligand to the bacteriochlorophyll (BChl) of the primary electron donor dimer M site (HisM200). With the loss of the histidine-coordinated Mg, one bacteriochlorophyll of the special pair was converted into a bacteriopheophytin (BPhe), and the primary donor became a heterodimer supermolecule. The crystals had dimensions 400 x 100 x 100 microm, belonged to space group P4(3)2(1)2, and were isomorphous to the ones reported earlier for the wild type (WT) strain. The structure was solved to a 2.5 A resolution limit. Electron-density maps confirmed the replacement of the histidine residue and the absence of Mg. Structural changes in the heterodimer mutant RC relative to the WT included the absence of the water molecule that is typically positioned between the M side of the primary donor and the accessory BChl, a slight shift in the position of amino acids surrounding the site of the mutation, and the rotation of the M194 phenylalanine. The cytochrome subunit was anchored similarly as in the WT and had no detectable changes in its overall position. The highly conserved tyrosine L162, located between the primary donor and the highest potential heme C(380), revealed only a minor deviation of its hydroxyl group. Concomitantly to modification of the BChl molecule, the redox potential of the heterodimer primary donor increased relative to that of the WT organism (772 mV vs. 517 mV). The availability of this heterodimer mutant and its crystal structure provides opportunities for investigating changes in light-induced electron transfer that reflect differences in redox cascades.

Racemic crystallography of synthetic protein enantiomers used to determine the X-ray structure of plectasin by direct methods.

We describe the use of racemic crystallography to determine the X-ray structure of the natural product plectasin, a potent antimicrobial protein recently isolated from fungus. The protein enantiomers L-plectasin and D-plectasin were prepared by total chemical synthesis; interestingly, L-plectasin showed the expected antimicrobial activity, while D-plectasin was devoid of such activity. The mirror image proteins were then used for racemic crystallization. Synchrotron X-ray diffraction data were collected to atomic resolution from a racemic plectasin crystal; the racemate crystallized in the achiral centrosymmetric space group P1 with one L-plectasin molecule and one D-plectasin molecule forming the unit cell. Dimer-like intermolecular interactions between the protein enantiomers were observed, which may account for the observed extremely low solvent content (13%-15%) and more highly ordered nature of the racemic crystals. The structure of the plectasin molecule was well defined for all 40 amino acids and was generally similar to the previously determined NMR structure, suggesting minimal impact of the crystal packing on the plectasin conformation.

Pentameric assembly of potassium channel tetramerization domain-containing protein 5.

We report the X-ray crystal structure of human potassium channel tetramerization domain-containing protein 5 (KCTD5), the first member of the family to be so characterized. Four findings were unexpected. First, the structure reveals assemblies of five subunits while tetramers were anticipated; pentameric stoichiometry is observed also in solution by scanning transmission electron microscopy mass analysis and analytical ultracentrifugation. Second, the same BTB (bric-a-brac, tramtrack, broad complex) domain surface mediates the assembly of five KCTD5 and four voltage-gated K(+) (Kv) channel subunits; four amino acid differences appear crucial. Third, KCTD5 complexes have well-defined N- and C-terminal modules separated by a flexible linker that swivels by approximately 30 degrees; the C-module shows a new fold and is required to bind Golgi reassembly stacking protein 55 with approximately 1 microM affinity, as judged by surface plasmon resonance and ultracentrifugation. Fourth, despite the homology reflected in its name, KCTD5 does not impact the operation of Kv4.2, Kv3.4, Kv2.1, or Kv1.2 channels.

Crystal structure of full-length KcsA in its closed conformation.

KcsA is a proton-activated, voltage-modulated K(+) channel that has served as the archetype pore domain in the Kv channel superfamily. Here, we have used synthetic antigen-binding fragments (Fabs) as crystallographic chaperones to determine the structure of full-length KcsA at 3.8 A, as well as that of its isolated C-terminal domain at 2.6 A. The structure of the full-length KcsA-Fab complex reveals a well-defined, 4-helix bundle that projects approximately 70 A toward the cytoplasm. This bundle promotes a approximately 15 degree bending in the inner bundle gate, tightening its diameter and shifting the narrowest point 2 turns of helix below. Functional analysis of the full-length KcsA-Fab complex suggests that the C-terminal bundle remains whole during gating. We suggest that this structure likely represents the physiologically relevant closed conformation of KcsA.

X-ray structure of native scorpion toxin BmBKTx1 by racemic protein crystallography using direct methods.

Racemic protein crystallography, enabled by total chemical synthesis, has allowed us to determine the X-ray structure of native scorpion toxin BmBKTx1; direct methods were used for phase determination. This is the first example of a protein racemate that crystallized in space group I41/a.

Simple host-guest chemistry to modulate the process of concentration and crystallization of membrane proteins by detergent capture in a microfluidic device.

This paper utilizes cyclodextrin-based host-guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-beta-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way.

X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers.

Chemical protein synthesis and racemic protein crystallization were used to determine the X-ray structure of the snow flea antifreeze protein (sfAFP). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. More facile crystal formation also occurred from a quasi-racemic mixture of d-sfAFP and l-Se-sfAFP, a chemical protein analogue that contains an additional -SeCH2- moiety at one residue and thus differs slightly from the true enantiomer. Multiple wavelength anomalous dispersion (MAD) phasing from quasi-racemate crystals was then used to determine the X-ray structure of the sfAFP protein molecule. The resulting model was used to solve by molecular replacement the X-ray structure of l-sfAFP to a resolution of 0.98 A. The l-sfAFP molecule is made up of six antiparallel left-handed PPII helixes, stacked in two sets of three, to form a compact brick-like structure with one hydrophilic face and one hydrophobic face. This is a novel experimental protein structure and closely resembles a structural model proposed for sfAFP. These results illustrate the utility of total chemical synthesis combined with racemic crystallization and X-ray crystallography for determining the unknown structure of a protein.

Toward chaperone-assisted crystallography: protein engineering enhancement of crystal packing and X-ray phasing capabilities of a camelid single-domain antibody (VHH) scaffold.

A crystallization chaperone is an auxiliary protein that binds to a target of interest, enhances and modulates crystal packing, and provides high-quality phasing information. We critically evaluated the effectiveness of a camelid single-domain antibody (V(H)H) as a crystallization chaperone. By using a yeast surface display system for V(H)H, we successfully introduced additional Met residues in the core of the V(H)H scaffold. We identified a set of SeMet-labeled V(H)H variants that collectively produced six new crystal forms as the complex with the model antigen, RNase A. The crystals exhibited monoclinic, orthorhombic, triclinic, and tetragonal symmetry and have one or two complexes in the asymmetric unit, some of which diffracted to an atomic resolution. The phasing power of the Met-enriched V(H)H chaperone allowed for auto-building the entire complex using single-anomalous dispersion technique (SAD) without the need for introducing SeMet into the target protein. We show that phases produced by combining SAD and V(H)H model-based phases are accurate enough to easily solve structures of the size reported here, eliminating the need to collect multiple wavelength multiple-anomalous dispersion (MAD) data. Together with the presence of high-throughput selection systems (e.g., phage display libraries) for V(H)H, the enhanced V(H)H domain described here will be an excellent scaffold for producing effective crystallization chaperones.

High-resolution structure of a self-assembly-competent form of a hydrophobic peptide captured in a soluble beta-sheet scaffold.

beta-Rich self-assembly is a major structural class of polypeptides, but still little is known about its atomic structures and biophysical properties. Major impediments for structural and biophysical studies of peptide self-assemblies include their insolubility and heterogeneous composition. We have developed a model system, termed peptide self-assembly mimic (PSAM), based on the single-layer beta-sheet of Borrelia outer surface protein A. PSAM allows for the capture of a defined number of self-assembly-like peptide repeats within a water-soluble protein, making structural and energetic studies possible. In this work, we extend our PSAM approach to a highly hydrophobic peptide sequence. We show that a penta-Ile peptide (Ile(5)), which is insoluble and forms beta-rich self-assemblies in aqueous solution, can be captured within the PSAM scaffold in a form capable of self-assembly. The 1.1-A crystal structure revealed that the Ile(5) stretch forms a highly regular beta-strand within this flat beta-sheet. Self-assembly models built with multiple copies of the crystal structure of the Ile(5) peptide segment showed no steric conflict, indicating that this conformation represents an assembly-competent form. The PSAM retained high conformational stability, suggesting that the flat beta-strand of the Ile(5) stretch primed for self-assembly is a low-energy conformation of the Ile(5) stretch and rationalizing its high propensity for self-assembly. The ability of the PSAM to "solubilize" an otherwise insoluble peptide stretch suggests the potential of the PSAM approach to the characterization of self-assembling peptides.

Structural basis for the function and inhibition of an influenza virus proton channel.

The M2 protein from influenza A virus is a pH-activated proton channel that mediates acidification of the interior of viral particles entrapped in endosomes. M2 is the target of the anti-influenza drugs amantadine and rimantadine; recently, resistance to these drugs in humans, birds and pigs has reached more than 90% (ref. 1). Here we describe the crystal structure of the transmembrane-spanning region of the homotetrameric protein in the presence and absence of the channel-blocking drug amantadine. pH-dependent structural changes occur near a set of conserved His and Trp residues that are involved in proton gating. The drug-binding site is lined by residues that are mutated in amantadine-resistant viruses. Binding of amantadine physically occludes the pore, and might also perturb the pK(a) of the critical His residue. The structure provides a starting point for solving the problem of resistance to M2-channel blockers.

The 1.38 A crystal structure of DmsD protein from Salmonella typhimurium, a proofreading chaperone on the Tat pathway.

The DmsD protein is necessary for the biogenesis of dimethyl sulphoxide (DMSO) reductase in many prokaryotes. It performs a critical chaperone function initiated through its binding to the twin-arginine signal peptide of DmsA, the catalytic subunit of DMSO reductase. Upon binding to DmsD, DmsA is translocated to the periplasm via the so-called twin-arginine translocation (Tat) pathway. Here we report the 1.38 A crystal structure of the protein DmsD from Salmonella typhimurium and compare it with a close functional homolog, TorD. DmsD has an all-alpha fold structure with a notable helical extension located at its N-terminus with two solvent exposed hydrophobic residues. A major difference between DmsD and TorD is that TorD structure is a domain-swapped dimer, while DmsD exists as a monomer. Nevertheless, these two proteins have a number of common features suggesting they function by using similar mechanisms. A possible signal peptide-binding site is proposed based on structural similarities. Computational analysis was used to identify a potential GTP binding pocket on similar surfaces of DmsD and TorD structures.

Synthetic antibodies for specific recognition and crystallization of structured RNA.

Antibodies that bind protein antigens are indispensable in biochemical research and modern medicine. However, knowledge of RNA-binding antibodies and their application in the ever-growing RNA field is lacking. Here we have developed a robust approach using a synthetic phage-display library to select specific antigen-binding fragments (Fabs) targeting a large functional RNA. We have solved the crystal structure of the first Fab-RNA complex at 1.95 A. Capability in phasing and crystal contact formation suggests that the Fab provides a potentially valuable crystal chaperone for RNA. The crystal structure reveals that the Fab achieves specific RNA binding on a shallow surface with complementarity-determining region (CDR) sequence diversity, length variability, and main-chain conformational plasticity. The Fab-RNA interface also differs significantly from Fab-protein interfaces in amino acid composition and light-chain participation. These findings yield valuable insights for engineering of Fabs as RNA-binding modules and facilitate further development of Fabs as possible therapeutic drugs and biochemical tools to explore RNA biology.

Structural basis for activation of the autoinhibitory C-terminal kinase domain of p90 RSK2.

The X-ray structure at 2.0-A resolution of the p90 ribosomal S6 kinase 2 C-terminal kinase domain revealed a C-terminal autoinhibitory alphaL-helix that was embedded in the kinase scaffold and determines the inactive kinase conformation. We suggest a mechanism of activation through displacement of the alphaL-helix and rearrangement of the conserved residue Glu500, as well as the reorganization of the T-loop into the active conformation.

Total synthesis by modern chemical ligation methods and high resolution (1.1 A) X-ray structure of ribonuclease A.

The total chemical synthesis of RNase A using modern chemical ligation methods is described, illustrating the significant advances that have been made in chemical protein synthesis since Gutte and Merrifield's pioneering preparation of RNase A in 1969. The identity of the synthetic product was confirmed through rigorous characterization, including the determination of the X-ray crystal structure to 1.1 Angstrom resolution.

Beta-strand flipping and slipping triggered by turn replacement reveal the opportunistic nature of beta-strand pairing.

We investigated how the register between adjacent beta-strands is specified using a series of mutants of the single-layer beta-sheet (SLB) in Borrelia OspA. The single-layer architecture of this system eliminates structural restraints imposed by a hydrophobic core, enabling us to address this question. A critical turn (turn 9/10) in the SLB was replaced with a segment with an intentional structural mismatch. Its crystal structure revealed a one-residue insertion into the central beta-strand (strand 9) of the SLB. This insertion triggered a surprisingly large-scale structural rearrangement: (i) the central strand (strand 9) was shifted by one residue, causing the strand to flip with respect to the adjacent beta-strands and thus completely disrupting the native side-chain contacts; (ii) the three-residue turn located on the opposite end of the beta-strand (turn 8/9) was pushed into its preceding beta-strand (strand 8); (iii) the register between strands 8 and 9 was shifted by three residues. Replacing the original sequence for turn 8/9 with a stronger turn motif restored the original strand register but still with a flipped beta-strand 9. The stability differences of these distinct structures were surprisingly small, consistent with an energy landscape where multiple low-energy states with different beta-sheet configurations exist. The observed conformations can be rationalized in terms of maximizing the number of backbone H-bonds. These results suggest that adjacent beta-strands "stick" through the use of factors that are not highly sequence specific and that beta-strands could slide back and forth relatively easily in the absence of external elements such as turns and tertiary packing.

Exploring the capacity of minimalist protein interfaces: interface energetics and affinity maturation to picomolar KD of a single-domain antibody with a flat paratope.

A major architectural class in engineered binding proteins ("antibody mimics") involves the presentation of recognition loops off a single-domain scaffold. This class of binding proteins, both natural and synthetic, has a strong tendency to bind a preformed cleft using a convex binding interface (paratope). To explore their capacity to produce high-affinity interfaces with diverse shape and topography, we examined the interface energetics and explored the affinity limit achievable with a flat paratope. We chose a minimalist paratope limited to two loops found in a natural camelid heavy-chain antibody (VHH) that binds to ribonuclease A. Ala scanning of the VHH revealed only three "hot spot" side chains and additional four residues important for supporting backbone-mediated interactions. The small number of critical residues suggested that this is not an optimized paratope. Using selection from synthetic combinatorial libraries, we enhanced its affinity by >100-fold, resulting in variants with Kd as low as 180 pM with no detectable loss of binding specificity. High-resolution crystal structures revealed that the mutations induced only subtle structural changes but extended the network of interactions. This resulted in an expanded hot spot region including four additional residues located at the periphery of the paratope with a concomitant loss of the so-called "O-ring" arrangement of energetically inert residues. These results suggest that this class of simple, single-domain scaffolds is capable of generating high-performance binding interfaces with diverse shape. More generally, they suggest that highly functional interfaces can be designed without closely mimicking natural interfaces.

High-affinity single-domain binding proteins with a binary-code interface.

High degrees of sequence and conformation complexity found in natural protein interaction interfaces are generally considered essential for achieving tight and specific interactions. However, it has been demonstrated that specific antibodies can be built by using an interface with a binary code consisting of only Tyr and Ser. This surprising result might be attributed to yet undefined properties of the antibody scaffold that uniquely enhance its capacity for target binding. In this work we tested the generality of the binary-code interface by engineering binding proteins based on a single-domain scaffold. We show that Tyr/Ser binary-code interfaces consisting of only 15-20 positions within a fibronectin type III domain (FN3; 95 residues) are capable of producing specific binding proteins (termed "monobodies") with a low-nanomolar K(d). A 2.35-A x-ray crystal structure of a monobody in complex with its target, maltose-binding protein, and mutation analysis revealed dominant contributions of Tyr residues to binding as well as striking molecular mimicry of a maltose-binding protein substrate, beta-cyclodextrin, by the Tyr/Ser binary interface. This work suggests that an interaction interface with low chemical diversity but with significant conformational diversity is generally sufficient for tight and specific molecular recognition, providing fundamental insights into factors governing protein-protein interactions.

Hydrophobic surface burial is the major stability determinant of a flat, single-layer beta-sheet.

Formation of a flat beta-sheet is a fundamental event in beta-sheet-mediated protein self-assembly. To investigate the contributions of various factors to the stability of flat beta-sheets, we performed extensive alanine-scanning mutagenesis experiments on the single-layer beta-sheet segment of Borrelia outer surface protein A (OspA). This beta-sheet segment consists of beta-strands with highly regular geometries that can serve as a building block for self-assembly. Our Ala-scanning approach is distinct from the conventional host-guest method, in that it introduces only conservative, truncation mutations that should minimize structural perturbation. Our results showed very weak correlation with experimental beta-sheet propensity scales, statistical beta-sheet propensity scales, or cross-strand pairwise correlations. In contrast, our data showed strong positive correlation with the change in buried non-polar surface area. Polar interactions including prominent Glu-Lys cross-strand pairs contribute marginally to the beta-sheet stability. These results were corroborated by results from additional non-Ala mutations. Taken together, these results demonstrate the dominant contribution of non-polar surface burial to flat beta-sheet stability even at solvent-exposed positions. The OspA single-layer beta-sheet achieves efficient hydrophobic surface burial without forming a hydrophobic core by a strategic placement of a variety of side-chains. These findings further suggest the importance of hydrophobic interactions within a beta-sheet layer in peptide self-assembly.