Domain association in immunoglobulin molecules. The packing of variable domains

We have analyzed the structure of the interface between VL and VH domains in three immunoglobulin fragments: Fab KOL, Fab NEW and Fab MCPC 603. About 1800 A2 of protein surface is buried between the domains. Approximately three quarters of this interface is formed by the packing of the VL and VH beta-sheets in the conserved "framework" and one quarter from contacts between the hypervariable regions. The beta-sheets that form the interface have edge strands that are strongly twisted (coiled) by beta-bulges. As a result, the edge strands fold back over their own beta-sheet at two diagonally opposite corners. When the VL and VH domains pack together, residues from these edge strands form the central part of the interface and give what we call a three-layer packing; i.e. there is a third layer composed of side-chains inserted between the two backbone side-chain layers that are usually in contact. This three-layer packing is different from previously described beta-sheet packings. The 12 residues that form the central part of the three observed VL-VH packings are absolutely or very strongly conserved in all immunoglobulin sequences. This strongly suggests that the structure described here is a general model for the association of VL and VH domains and that the three-layer packing plays a central role in forming the antibody combining site.     

An analysis of incorrectly folded protein models. Implications for structure predictions

Proteins with homologous amino acid sequences have similar folds and it has been assumed that an unknown three-dimensional structure can be obtained from a known homologous structure by substituting new side-chains into the polypeptide chain backbone, followed by relatively small adjustment of the model. To examine this approach of structure prediction and, more generally, to isolate the characteristics of native proteins, we constructed two incorrectly folded protein models. Sea-worm hemerythrin and the variable domain of mouse immunoglobulin K-chain, two proteins with no sequence homology, were chosen for study; the former is composed of a bundle of four alpha-helices and the latter consists of two 4-stranded beta-sheets. Using an automatic computer procedure, hemerythrin side-chains were substituted into the immunoglobulin domain and vice versa. The structures were energy-minimized with the program CHARMM and the resulting structures compared with the correctly folded forms. It was found that the incorrect side-chains can be incorporated readily into both types of structures (alpha-helices, beta-sheets) with only small structural adjustments. After constrained energy-minimization, which led to an average atomic co-ordinate shift of no more than 0.7 to 0.9 A, the incorrectly folded models arrived at potential energy values comparable to those of the correct structures. Detailed analysis of the energy results shows that the incorrect structures have less stabilizing electrostatic, van der Waals' and hydrogen-bonding interactions. The difference is particularly pronounced when the electrostatic and van der Waals' energy terms are calculated by modified equations that include an approximate representation of solvent effects. The incorrectly folded structures also have a significantly larger solvent-accessible surface and a greater fraction of non-polar side-chain atoms exposed to solvent. Examination of their interior shows that the packing of side-chains at the secondary structure interfaces, although corresponding to sterically allowed conformations, deviates from the characteristics found in normal proteins. The analysis of incorrectly folded structures has made it clear that the absence of bad non-bonded contacts, though necessary, is not sufficient to demonstrate the validity of model-built structures and that modeling of homologous structures has to be accompanied by a thorough quantitative evaluation of the results. Further, certain features that characterize native proteins are made evident by their absence in misfolded models.     

Repressor-operator interaction in the lac operon: III. Nuclear magnetic resonance observations with altered amino-terminal DNA binding domains

We have examined the N-terminal 56 amino acid fragment, the domain that can bind DNA independently, from 3-fluorotyrosine-substituted Escherichia coli lac repressor by ¹⁹F-nuclear magnetic resonance. The fragments or “headpieces” from four altered repressers missing each of the tyrosines in turn were examined in parallel. When the wild-type N-terminal fragment is titrated with a 36 base-pair lac operator DNA sequence, the ¹⁹F resonances undergo changes in their chemical shifts that are different from those changes when the N-terminal fragment is titrated with non-specific DNA fragments. By looking at these operator-induced changes as well as pH-dependent effects with all four altered N-terminal fragments, we show systematic correlations with the genetic data. The data lead us to conclude that upon operator DNA binding: (1) tyrosine 7 is displaced to a less polar environment and the higher than normal pK value of the phenolic OH group is decreased; (2) tyrosine 12 does not change much in either its mobility or environment; and (3) tyrosine 17 is involved, as suggested by the genetic data, when the headpiece forms a complex with operator DNA.     

Structure of ferricytochrome c' from Rhodospirillum molischianum at 1.67 A resolution

The structure of ferricytochrome c' from Rhodospirillum molischianum has been crystallographically refined to 1.67 A resolution using a combination of reciprocal space and restrained least-squares refinement methods. The final crystallographic R-factor for 30,533 reflections measured with I greater than sigma (I) between infinity and 1.67 A is 0.188. The final model incorporates 1944 unique protein atoms (of a total of 1972) together with 194 bound solvent molecules. The structure has been analysed with respect to its detailed conformational properties, secondary structural features, temperature factor behavior, bound solvent sites, and heme geometry. The asymmetric unit of the cytochrome c' crystal contains a dimer composed of chemically identical 128-residue polypeptide chains. Although the refined structure shows the monomers to be very similar, examination of the differences that do occur allows an evaluation of how different lattice contacts affect protein conformation and solvent binding. In particular, comparison of solvent binding sites in the two subunits allows identification of a common set that are not altered by lattice interactions. The preservation of these solvent interactions in different lattice environments suggests that they play a structural role in protein stabilization in solution. The refined structure additionally reveals some new features that relate to the ligand binding properties and unusual mixed-spin state character of cytochrome c'. Finally, comparison of the heme binding geometry in cytochrome c' and other structurally unrelated c-type cytochromes shows that two alternative, but sterically favorable, conformational variants occur among the seven examples examined.     

Characterization of crystals of a cytochrome oxidase (nitrite reductase) from Pseudomonas aeruginosa by x-ray diffraction and electron microscopy

Cytochrome oxidase from Pseudomonas aeruginosa has been crystallized from 2 m-ammonium sulfate. The crystals occur principally as thin diamond-shaped plates of space group P2₁2₁2 with unit cell dimensions of 92 Å × 115 Å × 76 Å. Determination of the density of glutaraldehyde-fixed, water-equilibrated crystals (1.167 g/cm³), coupled with the unit cell volume (804,000 ų), indicates that there is one subunit (~63,000 Mr) per asymmetric unit. X-ray diffraction data which were limited to 12 Å resolution due to small crystal size were obtained for the hk0 and 0kl zones using precession photography. Amplitude and phase data for the hk0, 0kl, and h0l zones were obtained from computer-based Fourier analysis of appropriate micrographs recorded from negatively stained microplates and thin sections of larger crystals using minimal beam electron microscopy. For crystals embedded in the presence of tannic acid it was possible to achieve 20 Å resolution which is comparable to the resolution achieved with negative staining of thin crystalline arrays. In addition, unstained electron diffraction on glutaraldehyde-fixed, glucose-stabilized plates was recorded to a resolution of 9 Å. The three-dimensional packing of the cytochrome oxidase dimer in the unit cell has been deduced from computer reconstructed images of the three principal projections along the crystallographic axes. The cytochrome oxidase dimer is located in the unit cell with the dimer axis coincident with a crystallographic 2-fold axis; thus within the resolution of the present data in projection (9 Å) the two subunits are identical, in agreement with biochemical evidence. The crystals have been prepared with the enzyme in the fully oxidized state and upon reduction a progressive cracking of the crystals is observed, possibly due to a conformational change dependent on the oxidation state of the heme iron.     

Four-stranded DNA structure and DNA base methylation in the mechanism of action of restriction endonucleases.

We examined the probability of short palindromec DNA sequences to occur as four-stranded structures held together in double-helical DNA by the additional hydrogen bonds postulated by McGavin (1971). The likeliness of the palindromes to be folded at their symmetry axes to allow the additional hydrogen bonding was considered using published physicochemical evidence and theoretical deductions. We deduced that both in vivo and in vitro the requirements may be met for duplex DNA folding which would approach palindrome complementary base bairs and thus allow the formation of the additional hydrogen bonds. However, we propose hydrogen bonding between guanine-cytosine base pairs to be different than that proposed by McGavin. Using CPK atom models we found that formation of the tertiary conformation already proposed by other authors and which we call the cage structure may be prevented or hindered by adenine, guanine or cytosine methylation. The available experimental data on recognition and cleavage site specificity of the Type 11 restriction endonucleases were confronted with the cage model as an alternative of the cruciform model and with the postulated effects of base methylation. The published data did not contradict the validity of the cage model and the role of base methylation in preventing the four-stranded palindrome structure. An applicability of the basic ideas of four-stranded DNA and base methylation effect to the mechanism of action of modification methylases and other restriction endonucleases was shortly discussed, but only tentative conclusions could be reached.     

Room temperature, low-field C-n.m.r. spectra of degraded carrageenans. Part II. On the specificity of the autohydrolysis reaction in kappa/iota and mu/nu structures

A kappa/iota carrageenan from Gigartina skottsbergii and a partially cyclized mu/nu carrageenan from Iridaea undulosa were submitted to autohydrolysis. The ¹³C-n.m.r. spectra of the degraded products give structural information on the polysaccharides and show clearly that besides the known splitting of 3,6-anhydrogalactosidic linkages, the linkage between - -galactose 2,6-disulphate and β- -galactose 4-sulphate is cleaved with similar specific reaction rate.     

Protein folding: evaluation of some simple rules for the assembly of helices into tertiary structures with myoglobin as an example

A computer program designed to fold a peptide chain consisting solely of helical segments and connecting links of known length is described and evaluated. This study is a detailed extension of certain aspects of the earlier work of Ptitsyn &; Rashin (1975). Possible interaction sites on the helices are sequence dependent and are calculated as described by Richmond &; Richards (1978) using probable changes in solvent contact area as a guide. The helices are then paired according to the list of potential sites, with each helix being paired at least once. The lists of pairings are then examined geometrically, each site having a defined dihedral helix axis angle, a specified inter-helix axis distance, and defined rotations, when required, about each helix axis. Two simplified filters are used: (1) lengths of connecting links must be equal to or greater than the end-to-end distances of the helices; and (2) non-paired helices must not collide. With myoglobin as a test example and only six of the eight helices being considered, a conformation space consisting of more than 3 × 10⁸ structures was surveyed. The two filters reduced the acceptable structure list to 121. Slight readjustment of the parameters in the filters would have reduced this to 20 structures. Of these 20, one closely resembles the actual distribution of helices in myoglobin. The possible utility and pitfalls of this approach as part of an overall protein folding program are discussed.     

A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA.

A deoxyribonuclease specific for methylated DNA was isolated from Diplococcus pneumoniae. The enzyme, an endonuclease, degrades DNA for Escherichia coli to fragments of average molecular weight about half a million; it forms discrete fragments from phage lambda DNA. Methyl-deficient E. coli DNA is not attacked, neither is DNA from Micrococcus radiodurans, which contains no methylated adenine or cytosine. Nor is DNA from D. pneumoniae or phage T7 attacked. However, DNA from M. radiodurans, D. pneumoniae, and T7 is attacked after methylation with and E. coli extract. Methylated T7 DNA is degraded to discrete fragments. Although the genetic transforming activity of normal DNA from D. pneumoniae is not affected by the enzyme, transforming activity of methylated DNA is destroyed. The enzyme is designated endonuclease R Dpn I. Under certain conditions another enzyme of complementary specificity can be isolated. This enzyme, designated endonuclease R Dpn II, produces a similar pattern of fragments from the DNA of T7 without prior methylation of the DNA. It also degrades normal DNA for D. pneumoniae. It is suggested that this pair of enzymes plays a role in some unknown control process, which would involve a large fraction of the specific base sequences that are methylated in E. coli DNA and are present but not methylated in DNA from other sources.     

Evolution of sex-ratio: Brief review with mathematical study of some simple novel models

Besides reviewing the unusual case of sex-ratio in the lemming and presenting alternative analyses of general models in which the shift in the usual sex-ratio 1:1 is determined by autosomal or sex-linked mutant alleles, three novel models are presented, in which the shift on the progeny sex-ratio depends on the number of copies of a mutant allele present in the parental pair. The analysis of these models with additive effects shows that: 1) autosomal mutations that alter the usual sex-ratio are eliminated from the population; 2) mutations occurring on the X chromosome lead to an evolutionary stable 1:1 sex-ratio only if the mutation favors the production of males; when the mutant allele favors the production of females, however, females will prevail in the population, with a frequency dependent impact on δ (the deviation from the usual 0.5 proportion) ; for most of the range of possible values of δ the stable but extraordinary sex-ratio will vary from 1 male : 1 female to 1 male : 3 females or 1 male : 2 females approximately depending whether the mutant allele is randomly inactivated or not.     

Search for producers of restriction endonucleases with a given specificity]

Six site-specific restriction endonucleases were isolated from Bacillus thuringiensis strains chosen of 58 strains ought purposefully for production of restriction enzymes. All six strains produce isoschisomers of Sau3A.     

Digestion of highly modified bacteriophage DNA by restriction endonucleases.

The ability of thirty Type II restriction endonucleases to cleave five different types of highly modified DNA has been examined. The DNA substrates were derived from relatively large bacteriophage genomes which contain all or most of the cytosine or thymine residues substituted at the 5-position. These substituents were a proton (PBS1 DNA), a hydroxymethyl group (SP01 DNA), a methyl group (XP12 DNA), a glucosylated hydroxymethyl group (T4 DNA), or a phosphoglucuronated, glucosylated 4,5-dihydroxypentyl group (SP15 DNA). Although PBS1 DNA and SP01 DNA were digested by most of the enzymes, they were cleaved much more slowly than was normal DNA by many of them. 5-Methylcytosine-rich XP12 DNA and the multiply modified T4 and SP15 DNAs were resistant to most of these endonucleases. The only enzyme that cleaved all five of these DNAs was TaqI, which fragmented them extensively.     

Cleavage map of BK virus DNA with restriction endonucleases MboI and HaeIII.

Specific cleavage of BK virus (MM) DNA with restriction endonuclease MboI gives rise to 10 fragments. Two techniques were used to determine the location of these fragments on the viral genome with respect to the three known sites for HindIII cleavage. In the first method, reciprocal digestion, individual MboI fragments were digested with HindIII and individual HindIII fragments were digested with MboI. In the second method, single-end 32P-labeled HindIII subfragments were partially digested with MboI, and then the sizes of the radioactive partial products were used to deduce the nearest neighboring fragment. Information from these two methods is more than adequate to map all the MboI enzyme sites. Cleavage of BK virus (MM) DNA with restriction enzyme HaeIII produces 21 fragments. With the aid of the same two methods, these fragments have also been ordered with respect to the known map locations of the HindIII and MboI sites.     

Analysis of the activity of Micrococcus luteus endonucleases with respect to gamma-irradiated DNA]

Endonucleases from Micrococcus luteus that induce single-strand breaks in gamma-irradiated DNA have been separated chromatographycally into two groups. The first group involves two different enzymes: AP-endonuclease II (mol. weight 30 000) and AP, UV-endonuclease I (mol. weight 15 000) that recognize alkali-labile lesions in gamma-irradiated DNA and apurinic sites in DNA heated at 70 degrees C, pH 6.08 AP-endonuclease II in cooperation with DNA polymerase from M. luteus and T4 phage-induced polynucleotide ligase is capable of carrying out in vitro complete excision repair of alkali-labile lesins in gamma-irradiated DNA. The second group involves gamma-endonucleases X and Y that act on alkalistable gamma-ray lesions. gamma-endonucleases X and Y can be separated by chromatography on DEAE-cellulose but possess similar properties. Activity of gamma-endonucleases toward gamma-irradiated DNA is inhibited by only heavily UV-irradiated DNA (15 000 ergs/mm2). The data are consistent with the hypothesis that gamma-endonucleases are specific for thymine glycols (t' and tUV) in UV- and gamma-irradiated DNA.     

Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity

Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity. MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize. The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure. The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before. This study provides evidence that MABS is a promising technique for generation of REs with new specificities.     

Action of restriction endonucleases on transforming DNA of Haemophilus influenzae.

Cleavage of DNA from Haemophilus influenzae with restriction endonucleases caused inactivation of transforming ability to an extent that depended on the genetic marker and the enzyme. The rate of inactivation, but not the final level of survival, depended on the concentration of enzyme in the restriction digest. In general, the greatest extent of inactivation of transforming activity was obtained with endonucleases that are known to produce the shortest fragments. We electrophoresed restriction digests of H. influenzae DNA in agarose gels and assayed transforming activity of DNA extracted from gel slices. In this way, we determined the lengths of restriction fragments that contain genetic markers of H. influenzae. For the marker that we studied most thoroughly (nov), the shortest restriction fragment that possessed detectable transforming activity was a 0.9-kilobase pair fragment produced by endonuclease R . PstI. The shortest marker-bearing restriction fragment that retained substantial transforming activity (50% of value for undigested DNA) was a 2.1-kilobase pair EcoRI fragment bearing the kan marker. Among marker-bearing restriction fragments 1 to 4 kilobase pairs in length, survival of transforming activity varied 10,000-fold. We relate these observations to the recent findings by Sisco and Smith (Proc. Natl. Acad. Sci. U.S.A. 76:972-976, 1979) that efficient entry of DNA into competent H. influenzae cells appears to require the presence of a recognition sequence that is scattered throughout the Haemophilus genome in many more copies than in unrelated genomes.