Apostolos G. Gittis

High Apparent Dielectric Constants in the Interior of a Protein Reflect Water Penetration

A glutamic acid was buried in the hydrophobic core of staphylococcal nuclease by replacement of Val-66. Its pK(a) was measured with equilibrium thermodynamic methods. It was 4.3 units higher than the pK(a) of Glu in water. This increase was comparable to the DeltapK(a) of 4.9 units measured previously for a lysine buried at the same location. According to the Born formalism these DeltapK(a) are energetically equivalent to the transfer of a charged group from water to a medium of dielectric constant of 12. In contrast, the static dielectric constants of dry protein powders range from 2 to 4. In the crystallographic structure of the V66E mutant, a chain of water molecules was seen that hydrates the buried Glu-66 and links it with bulk solvent. The buried water molecules have never previously been detected in >20 structures of nuclease. The structure and the measured energetics constitute compelling and unprecedented experimental evidence that solvent penetration can contribute significantly to the high apparent polarizability inside proteins. To improve structure-based calculations of electrostatic effects with continuum methods, it will be necessary to learn to account quantitatively for the contributions by solvent penetration to dielectric effects in the protein interior.

Experimental measurement of the effective dielectric in the hydrophobic core of a protein

The dielectric inside a protein is a key physical determinant of the magnitude of electrostatic interactions in proteins. We have measured this dielectric phenomenologically, in terms of the dielectric that needs to be used with the Born equation in order to reproduce the observed pKa shifts induced by burial of an ionizable group in the hydrophobic core of a protein. Mutants of staphylococcal nuclease with a buried lysine residue at position 66 were engineered for this purpose. The pKa values of buried lysines were measured by difference potentiometry. The extent of coupling between the pKa and the global stability of the protein was evaluated by measuring pKa values in hyperstable forms of nuclease engineered to be 3.3 or 6.5 kcal mol-1 more stable than the wild type. The crystallographic structure of one mutant was determined to describe the environment of the buried lysine. The dielectrics that were measured range from 10 to 12. Published pKa values of buried ionizable residues in other proteins were analyzed in a similar fashion and the dielectrics obtained from these values are consistent with the ones measured in nuclease. These results argue strongly against the prevalent use of dielectrics of 4 or lower to describe the dielectric effect inside a protein in structure-based calculations of electrostatic energies with continuum dielectric models.

In a staphylococcal nuclease mutant the side-chain of a lysine replacing valine 66 is fully buried in the hydrophobic core

The crystal structure of the staphylococcal nuclease mutant V66K, in which valine 66 is replaced by lysine, has been solved at 1.97 A resolution. Unlike lysine residues in previously reported protein structures, this residue appears to bury its side-chain in the hydrophobic core without salt bridging, hydrogen bonding or other forms of electrostatic stabilization. Solution studies of the free energy of denaturation, delta GH2O, show marked pH dependence and clearly indicate that the lysine residue must be deprotonated in the folded state. V66K is highly unstable at neutral pH but only modestly less stable than the wild-type protein at high pH. The pH dependence of stability for V66K, in combination with similar measurements for the wild-type protein, allowed determination of the pKa values of the lysine in both the denatured and native forms. The epsilon-amine of this residue has a pKa value in the denatured state of 10.2, but in the native state it must be 6.4 or lower. The epsilon-amine is thus deprotonated in the folded molecule. These values enabled an estimation of the epsilon-amines relative change in free energy of solvation between solvent and the protein interior at 5.1 kcal/mol or greater. This implies that the value of the dielectric constant of the protein interior must be less than 12.8. Lysine is usually found with the methylene groups of its side-chain partly buried but is nevertheless considered a hydrophilic surface residue. It would appear that the high pKa value of lysine, which gives it a positive charge at physiological pH, is the primary reason for its almost exclusive confinement to the surface proteins. When deprotonated, this amino acid type can be fully incorporated into the hydrophobic core.

One-step evolution of a dimer from a monomeric protein

Deletion of six amino acids in a surface loop transforms staphylococcal nuclease from a monomeric protein into a very stable dimer (Kd<1×10−8M). A 2 Å X-ray crystal structure of the dimer (R=0.176) shows that the carboxy-terminal α-helix has been stripped from its normal position in one monomer and is now incorporated into the equivalent position on the adjoining monomer. This swapping creates an association interface of 2900 Å2. A second, smaller interface of 460 Å2 is also formed. The spontaneous exchange or swapping of secondary structural elements provides a simple pathway for the formation of large, stable protein/protein interfaces and may play an important role in the evolution of oligomeric proteins.

A Compact RNA Tertiary Structure Contains a Buried Backbone-K+ Complex

The structure of a 58 nucleotide ribosomal RNA fragment buries several phosphate groups of a hairpin loop within a large tertiary core. During refinement of an X-ray crystal structure containing this RNA, a potassium ion was found to be contacted by six oxygen atoms from the buried phosphate groups; the ion is contained completely within the solvent-accessible surface of the RNA. The electrostatic potential at the ion chelation site is unusually large, and more than compensates for the substantial energetic penalties associated with partial dehydration of the ion and displacement of delocalized ions. The very large predicted binding free energy, approximately -30 kcal/mol, implies that the site must be occupied for the RNA to fold. These findings agree with previous studies of the ion-dependent folding of tertiary structure in this RNA, which concluded that a monovalent ion was bound in a partially dehydrated environment where Mg2+ could not easily compete for binding. By compensating the unfavorable free energy of buried phosphate groups with a chelated ion, the RNA is able to create a larger and more complex tertiary fold than would be possible otherwise.

Cytosolic domain of the human mitochondrial fission protein fis1 adopts a TPR fold

Fis1 is an integral membrane protein that acts in the fission of mitochondria by controlling the assembly, membrane distribution, and function of the mitochondrial fission machinery. Here we report the 2.0 A resolution crystal structure of the cytosolic domain of human Fis1. The structure reveals an antiparallel array of α-helices homologous to tetratricopeptide repeat (TPR) proteins. Structure-based sequence alignments of Fis1 uncovered two divergent TPR motifs; the first TPR motif differs from the TPR consensus sequence by a three-residue insertion in a loop that may be important for function. These TPR helices create an amphiphilic, concave surface that can accommodate a helix or, possibly, an extended segment. Indeed, this putative binding surface mediates homodimer formation of Fis1 in the crystal. The structure of Fis1 provides insight into the architecture of the proposed binding interactions that mediate mitochondrial fission.

The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

ABSTRACT The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.

Crystallographic study of hydration of an internal cavity in engineered proteins with buried polar or ionizable groups.

Although internal water molecules are essential for the structure and function of many proteins, the structural and physical factors that govern internal hydration are poorly understood. We have examined the molecular determinants of internal hydration systematically, by solving the crystal structures of variants of staphylococcal nuclease with Gln-66, Asn-66, and Tyr-66 at cryo (100 K) and room (298 K) temperatures, and comparing them with existing cryo and room temperature structures of variants with Glu-66, Asp-66, Lys-66, Glu-92 or Lys-92 obtained under conditions of pH where the internal ionizable groups are in the neutral state. At cryogenic temperatures the polar moieties of all these internal side chains are hydrated except in the cases of Lys-66 and Lys-92. At room temperature the internal water molecules were observed only in variants with Glu-66 and Tyr-66; water molecules in the other variants are probably present but they are disordered and therefore undetectable crystallographically. Each internal water molecule establishes between 3 and 5 hydrogen bonds with the protein or with other internal water molecules. The strength of interactions between internal polar side chains and water molecules seems to decrease from carboxylic acids to amides to amines. Low temperature, low cavity volume, and the presence of oxygen atoms in the cavity increase the positional stability of internal water molecules. This set of structures and the physical insight they contribute into internal hydration will be useful for the development and benchmarking of computational methods for artificial hydration of pockets, cavities, and active sites in proteins.

Molecular insights into the binding of coenzyme F420 to the conserved protein Rv1155 from Mycobacterium tuberculosis

Coenzyme F420 is a deazaflavin hydride carrier with a lower reduction potential than most flavins. In Mycobacterium tuberculosis (Mtb), F420 plays an important role in activating PA‐824, an antituberculosis drug currently used in clinical trials. Although F420 is important to Mtb redox metabolism, little is known about the enzymes that bind F420 and the reactions that they catalyze. We have identified a novel F420‐binding protein, Rv1155, which is annotated in the Mtb genome sequence as a putative flavin mononucleotide (FMN)‐binding protein. Using biophysical techniques, we have demonstrated that instead of binding FMN or other flavins, Rv1155 binds coenzyme F420. The crystal structure of the complex of Rv1155 and F420 reveals one F420 molecule bound to each monomer of the Rv1155 dimer. Structural, biophysical, and bioinformatic analyses of the Rv1155–F420 complex provide clues about its role in the bacterium.

The duplication of an eight-residue helical stretch in Staphylococcal nuclease is not helical: a model for evolutionary change.

A common method of evolutionary change is gene duplication, followed by other events that lead to new function, decoration of folds, oligomerization, or other changes. As part of a study on the potential for evolutionary change created by duplicated sequences, we have carried out a crystallographic study on a mutant of Staphylococcal nuclease in which residues 55–62 have been duplicated in a wild‐type variant termed PHS. In the parental protein (PHS) these residues form the first two turns of a helix running from residue 54 to 68 (hereafter designated as helix I). The crystal structure of the mutant is very similar to that of the parental, with helix I being unaltered. The duplicated residues are accommodated by expanding an existing loop N‐terminal to helix I. In addition, circular dichroism (CD) studies have been carried out on a parental peptide containing helix I with six flanking residues at each terminus (residues 48–74) and on the same peptide expanded by the duplication, as a function of 2,2,2‐trifluoroethanol (TFE) concentration. Each peptide possesses only modest helical propensity in solution. Our data, which is different from what was observed in T4 lysozyme, show that the conformation of the duplicated sequence is determined by a balance of sequential and longer‐range effects. Thus duplicating sequence need not mean duplicating structure. Proteins 2000;40:465–472.