Change in the crystal packing of soybean ß-amylase mutants substituted at a few surface amino acid residues

You-Na Kang, Motoyasu Adachi, Bunzo Mikami1 and Shigeru Utsumi

Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

1 To whom correspondence should be addressed. e-mail: mikami{at}kais.kyoto-u.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In spite of the high similarity of amino acid sequence and three-dimensional structure between soybean ß-amylase (SBA) and sweet potato ß-amylase (SPB), their quaternary structure is quite different, being a monomer in SBA and a tetramer in SPB. Because most of the differences in amino acid sequences are found in the surface region, we tested the tetramerization of SBA by examining mutations of residues located at the surface. We designed the SBA tetramer using the SPB tetramer structure as a model and calculating the change of accessible surface area ({Delta}ASA) for each residue in order to select sites for the mutation. Two different mutant genes encoding SB3 (D374Y/L481R/P487D) and SB4 (K462S added to SB3), were constructed for expression in Escherichia coli and the recombinant proteins were purified. They existed as a monomer in solution, but gave completely different crystals from the native SBA. The asymmetric unit of the mutants contains four molecules, while that of native SBA contains one. The interactions of the created interfaces revealed that there were more intermolecular interactions in the SB3 than in the SB4 tetramer. The substituted charged residues on the surface are involved in interactions with adjacent molecules in a different way, forming a new crystal packing pattern.

Keywords: accessible surface area/crystal packing/crystal structure/soybean ß-amylase/tetramer


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ß-Amylase ({alpha}-1,4-glucan maltohydrolase; EC 3.2.1.2) catalyzes the liberation of ß-anomeric maltose from the non-reducing ends of starch and glycogen (Thoma et al., 1971Go). This enzyme occurs in higher plants and some microorganisms. Although amino acid sequences of ß-amylase from several sources show distinct conserved regions, the sequence identity is only ~30% between plant and bacterial ß-amylases. The bacterial ß-amylases have an additional C-terminal domain compared with the plant ß-amylases, which is referred to as the starch-binding domain (Nanmori, 1988Go; Pujadas et al., 1996Go).

Many plant ß-amylases have been cloned and sequenced from soybean, barley, rye, Arabidopsis thaliana and sweet potato (Kreis et al., 1987Go; Monroe et al., 1991Go; Rorat et al., 1991Go; Yoshida and Nakamura, 1991Go; Totsuka and Fukazawa, 1993Go). They have similar properties in terms of amino acid sequence, molecular mass (50–60 kDa), optimum pH and so on, except for the homotetramer structure of sweet potato ß-amylase (SPB). Among the many ß-amylases of plant origin, the soybean ß-amylase (SBA) and SPB show 67% amino acid sequence identity (Yoshida and Nakamura, 1991Go). In spite of the high sequence and structural similarity, SBA and SPB show a remarkable difference in their quaternary structure. While SBA is a monomer of 495 amino acid residues, SPB is a homotetramer of 498x4 amino acid residues. SPB is a very stable tetrameric enzyme, and the dissociation to a monomer is not easy under mild conditions. In order to obtain an active SPB monomer, Ahn et al. (Ahn et al., 1989Go) modified the tetramer SPB with periodate-oxidized soluble starch or maltohexaose at pH 9.7. They could improve the yield and the specific activity of the monomer by using {alpha}-cyclodextrin to protect the active site of SPB (Ahn et al., 1990aGo,b).

The recombinant SBA revealed almost the same properties and structure as the natural SBA (Adachi et al., 1998Go). The structural analysis of SBA revealed that it is composed of a (ß/{alpha})8 core domain (residues 13–91, 150–181, 238–296 and 325–442), a smaller lobe made up of three long loops [L3 from ß-strand ß3 (residues 92–149), L4 from ß4 (residues 182–257) and L5 (residues 297–324) of the (ß/{alpha})8 barrel], and a long C-terminal loop region formed by residues 443–495 (Mikami et al., 1993Go; Mikami, 1994Go). Two maltose molecules bind in tandem in the active site pocket of SBA occupying subsites –2 to –1 and +1 to +2, respectively (Mikami et al., 1993Go; Davies et al., 1997Go), the two catalytic residues, Glu186 and Glu380, being located between subsites –1 and +1. The open–closed conformation of the flexible loop (residues 96–103) was found to play a key role in the catalytic reaction (Mikami et al., 1994Go).

The three-dimensional structure of tetrameric SPB was solved by molecular replacement methods using the SBA structure as a search model at 2.3 Å by Cheong et al. (Cheong et al., 1995Go). The overall structures of SBA and SPB are very similar to each other, as demonstrated by the fact the root mean square deviation (r.m.s.d.) for 487 equivalent C{alpha} atoms of the two structures is only 0.96 Å (Cheong et al., 1995Go). Cheong et al. suggested that out of two putative tetramer models of SPB, case I and case II, the former is more likely than the latter. Interfaces of the tetramer structure of case I can be divided into two sections. One type is mainly formed by the loop L'7, the N-terminal region, and a part of the C-terminal region near residue number 480. Another type of interaction is composed of a part of the C-terminal long loop and the segment containing the C-terminal end of {alpha}4 and the loop L'4 (Cheong et al., 1995Go).

Most of the differences in amino acid residues between SBA and SPB are observed on the surface of the subunit structure. While the first five or more residues from the N-terminus are often not found in the crystal structure in SBA because of high flexibility, the N-terminus of SPB tetramer is engaged in the tetramer formation making intermolecular interactions with the C-terminal residues of the neighboring subunits. Surface amino acid residues are very important because they can interact with adjacent molecules or monomers, which means that the mutations of some critical amino acid residues located on the surface may affect both the local and the overall structure of the proteins by interacting with the neighboring molecules. Conversion of SBA from monomer to tetramer will help us to understand the molecular evolution of ß-amylase and to design a ß-amylase with improved stability (Thoma et al., 2000Go; Canals et al., 2001Go).

Here, we report on our efforts to change the monomeric SBA structure to a tetramer based on the SPB structure by the mutation of a few amino acid residues distributed on the surface. To determine the residues to be mutated, we considered residues that were most likely to be responsible for the tetramer structure in SPB, and compared these with the corresponding residues of SBA in terms of the accessible surface area (ASA). Remarkable changes of crystal packing were found in the crystal structures of the resulting mutants.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Comparison and identification of SBA and SPB sequences responsible for tetramerization

To determine the mutation sites for designing the SBA tetramer, we composed a predicted tetramer model of SBA (tSBA) based on the SPB tetramer by superimposing the former on the latter. The ASA was calculated for each isolated subunit (monomer) and the tetramer by the NACCESS program, which is an implementation of the method of Lee and Richards (Lee and Richards, 1971Go) algorithm developed by Hubbard et al. (Hubbard et al., 1991Go). The ASA is defined by rolling a probe of a given size around a van der Waals surface. The radius of a water molecule rolled over the surface of the molecular model was 1.4 Å. If a certain residue participates in the intersubunit interaction and thus can be an interface of the oligomer, the ASA at that residue in the oligomer state will be significantly decreased compared with the ASA at that residue in the monomer state. Therefore, the change of the ASA ({Delta}ASA, the subtraction of the ASA of the tetramer from the ASA of the monomer) at each residue on tetramerization can provide information about the residues that will be at interfaces during tetramer formation. On the basis of ASA calculations, the interface residues were defined as those residues with side chains possessing a {Delta}ASA >1 Å2 upon tetramer formation (Janin et al., 1988Go; Jones and Thornton, 1996Go). Next, we calculated the ratio of {Delta}ASA of SPB at each site ({Delta}ASASPB) to {Delta}ASA of tSBA ({Delta}ASASBA) and considered large values of {Delta}ASASPB/{Delta}ASASBA for mutation sites.

Construction and purification of SB3 and SB4

Plasmid DNA extraction, ligation and gel extraction were performed as described by Sambrook et al. (Sambrook et al., 1989Go). Oligonucleotides were purchased from Takara, Japan. The mutants were prepared by PCR, using cloned Pfu polymerase (Takara, Japan). The correct sequence of the two mutant genes was confirmed by DNA sequencing. Mutant protein substituted at three different sites of D374Y/L481R/P487D was given the name ‘SB3’, and at four sites altered by adding the K462S mutation to the SB3 mutant was called ‘SB4’. The expression plasmids pKSB3 and pKSB4 were transformed into Escherichia coli strain JM105. Cells of E.coli strain JM105 harboring pKSB3 and pKSB4 were grown in LB medium, supplemented with ampicillin (0.1 mg/ml) at 37°C. At an A600 of 0.8, the medium was cooled to 18°C, and isopropyl-ß-D-thiogalactopyranoside was added to a final concentration of 1 mM, after which cells were cultured for 40 h at 18°C. The cells were harvested by centrifugation and suspended in 300 ml of 0.1 M sodium phosphate buffer (pH 7.0) containing 5 mM ethylenediamine-N,N,N',N'-tetraacetic acid, disodium salt, dihydrate (EDTA), 0.1 M NaCl and 1 mM phenylmethylsulfonyl fluoride. Cells were then disrupted by sonication at 4°C.

After centrifugation at 6800 g for 20 min, the supernatant was collected and 2-mercaptoethanol (2-ME) was added to a final concentration of 20 mM. The resultant crude extract was then fractionated by 65% saturation with ammonium sulfate. The precipitated protein was diluted to 30% saturation by adding buffer and centrifuged to collect the supernatant. This supernatant was applied to a butyl-Toyopearl column (4x25 cm) previously equilibrated with 50 mM sodium phosphate buffer (pH 6.5) containing 5 mM EDTA, 20 mM 2-ME and 30% saturated ammonium sulfate. Proteins were eluted with a descending gradient of ammonium sulfate ranging from 30 to 0%. The fractions containing ß-amylase activity were pooled and dialyzed against 50 mM sodium acetate buffer at pH 4.8 containing 1 mM EDTA and 20 mM 2-ME. After dialysis, the protein solution was applied to an SP-Sepharose column (2x25 cm) previously equilibrated with 50 mM sodium acetate buffer (pH 4.8) containing 1 mM EDTA and 20 mM 2-ME. Proteins were eluted with a pH gradient of 50 mM sodium acetate buffer from 4.8 to 7.0. The active fractions were collected and applied to a Mono S column (1.5x10 cm) under the same conditions as described for the SP-Sepharose column. The purified mutant proteins were concentrated to 10 mg/ml by ultrafiltration using a Millipore UFV 4BTC25 (MWCO 10 000) and confirmed by SDS–PAGE (Laemmli, 1970Go), exhibiting a single band with a molecular weight of 56 000 Da.

Assay methods

ß-Amylase activity was measured in 0.1 M acetate buffer, pH 5.4 for the hydrolysis of amylopectin at 37°C, according to the Bernfeld method, with a slight modification as described in the previous report (Bernfeld, 1955Go; Adachi et al., 1998Go). One unit was defined as the activity releasing 1 µmol of maltose for 1 min.

Dynamic light scattering measurement

To confirm the average molecular weight of SB3 and SB4 in solution, dynamic light scattering (DLS) measurements were carried out on a DynaPro-801TM Molecular Sizing Instrument (Protein Solutions, Inc.) at 20°C. A 1 mg/ml concentration of mutant proteins in 0.1 M sodium acetate buffer pH 5.4, containing 18 mM 2-ME and 1 mM EDTA was used for the measurements. All samples were filtered through a 0.02 µm Whatman Anodisc prior to analysis. The average hydrodynamic radius (RH) and molecular weight were determined by the mean of at least 20 DLS measurements. Data analysis was performed using the Dynals version 5.0 software package.

Crystallization

Purified SB4 and SB3 were crystallized using the hanging-drop vapor diffusion method under the same conditions as for the native SBA (Mikami et al., 1993Go). The solution in the crystallization drop was prepared on a silanized cover slip by mixing 5 µl of a 10 mg/ml protein solution with 5 µl of a reservoir solution containing 45–50% ammonium sulfate, 1 mM EDTA, 18 mM 2-ME in 0.1 M sodium acetate buffer (pH 5.4). Droplets were equilibrated against 1ml of the bottom solution at 4°C.

Data collection and refinement

The crystal data of SB3 were collected at a resolution up to 2.1 Å, using CuK{alpha} radiation ({lambda} = 1.5418 Å) with a Bruker Hi-Star area detector coupled to a MAC Science M18XHF rotating-anode generator. The collected data were processed with the SADIE and SAINT software packages (Bruker). In the case of SB4, the diffraction data were collected at a resolution of up to 2.1 Å, using an Oxford PX210 CCD detector system at beam line BL44XU (Beam Line for Macromolecule Assembles, Institute for Protein Research, Osaka University) at SPring-8 (Hyogo, Japan). The data collection was carried out at 100 K under a nitrogen gas stream. The collected images were processed with the program d*TREK (Pflugrath, 1999Go).

The molecular replacement method was used to solve the structures of SB3 and SB4. The model of native SBA complexed with 200 mM maltose, refined to an R-value of 0.149 at 1.9 Å (RCSB Protein Data Bank, accession No. 1BYB) was used as a search model. Model building was performed using the graphics program TURBO-FRODO (AFMB-CNRS, France) on a Silicon Graphics Octane computer, and the refinement calculations were carried out with the program CNS (Brüger et al., 1998Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Determination of mutation sites for SBA tetramerization based on the SPB structure

Figure 1a shows the superimposition of the SBA structure onto that of SPB. The r.m.s.d. for 487 C{alpha} atoms between SBA and SPB was 0.96 Å. SPB has two types of interface within the tetramer, i.e., interface I (between subunits A and B, and between subunits C and D) and II (between subunits A and D, and between subunits B and C). The number and strength of the intersubunit interactions are much higher at interface I than at interface II. Cheong et al. suggested that if this model is correct, the SPB tetramer might be formed by a two-step process. In the first step, a dimer is formed creating interface I, and then two dimer molecules interact with each other, creating interface II, finally making a tetramer (Cheong et al., 1995Go). If the interactions at interface I are strong enough to form a stable dimer between two molecules, then two dimers formed in this manner will easily manage to make interactions between themselves at the next step. For this reason, we first focused on the interactions at interface I.



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Fig. 1. (a) Stereo image of the superimposition of the SPB tetramer and the designed SBA tetramer (tSBA). The structure of SPB is shown in yellow; that of tSBA is shown in blue. tSBA was designed based on the SPB tetramer structure. There are two interfaces in SPB, interface I between subunits A and B (also C and D) indicated by a red box, and interface II between subunits A and D (also B and C) shown in black. (b) Amino acid sequence alignment of SPB and SBA at interfaces I and II. The interface residues in SPB and SBA are indicated by bold and italics, respectively. The residues that actively participated in the intersubunit interaction of SPB are indicated in green. The reversible arrow means that there are reciprocal intersubunit interactions between the subunits. The vertical arrows point to identical sequence parts. Based on the comparison of the {Delta}ASA and the residue distance at the intersubunit between SPB and tSBA, four mutation sites were determined. Three are in interface I, D374Y, L481R and P487D, and the other one is in interface II, K462S.

 
Figure 1b shows the amino acid alignment of SPB and tSBA at interfaces I and II. All residue numbers follow the SBA sequence in the alignment. Based on the high {Delta}ASASPB/{Delta}ASASBA value, three mutation sites, D374Y, L481R and P487D, located on interface I of the SPB tetramer and predicted SBA tetramer, were determined to be involved in the creation of interface I.

The distance between the residues at the predicted interface was also considered as well as the ratio of {Delta}ASA between SPB and tSBA. In the simulated tetramer interface of monomeric SBA obtained from structural superimposition with SPB, K462 was in severe conflict with its partner residue in the other subunit, creating interface II when undergoing teramerization. Thus, it was changed to serine, corresponding to SPB. The K462S mutation was added to the three already present in SB3, and the resulting mutant was called SB4.

Enzymatic parameters and dynamic light scattering measurements of SB3 and SB4

The kinetic parameters of SB3 and SB4 mutant proteins for the amylopectin hydrolysis at 37°C are summarized in Table I. Both mutant proteins exhibited almost the same kcat and Km values as the wild-type enzyme indicating that these mutations did not affect the catalytic mechanism of the enzyme. Table I also shows the hydrodynamic radius (RH) and the calculated molecular weight of the mutant proteins confirmed by the dynamic light scattering experiment. Whereas the molecular weight of wild-type SBA is 56 kDa, the observed average RH for SB3 was 3.150 nm, corresponding to a molecular weight of 49.33 kDa and for SB4, they were 3.199 nm and 51.15 kDa, respectively, which indicate two mutant proteins exist as a monomer in solution.


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Table I. Kinetic parameters and molecular weights of wild-type and mutant SBA
 
Crystallization and X-ray data collection

The crystals of SB3 and SB4 were obtained under the same conditions as those of the wild-type enzyme within 2–3 weeks. The mutants had nearly identical crystal forms (Figure 2). While the crystal system of the wild-type SBA is trigonal (P3121) with cell dimensions of a = b = 86.4 Å and c = 144.0 Å, the SB3 and SB4 crystal system is triclinic, belonging to the space group P1 with unit cell dimensions of a = 75.100 Å, b = 78.140 Å, c = 87.944 Å, {alpha} = 89.926°, ß = 89.878°, {gamma} = 90.075° for SB3, and a = 75.463 Å, b = 78.801 Å, c = 88.560 Å, {alpha} = 89.921°, ß = 90.209°, {gamma} = 89.902° for SB4.



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Fig. 2. Crystals of (a) wild-type SBA, (b) SB3 (D374Y/L481Y/P487D) and (c) SB4 (SB3 + K462S).

 
The X-ray data collection and refinement statistics are summarized in Table II. The final model of SB3 consisted of 490x4 amino acid residues, 24 sulfate ions and 1461 water molecules with an R-factor of 18.26% (free R =24.08%) for 93 363 reflections of F > 0{sigma}(F) between the 10 and 2.1 Å resolution. In the case of SB4, the asymmetric unit contained 490x4 amino acid residues, 20 sulfate ions and 1005 water molecules with an R-factor of 20.01% (free R = 23.45%) for 112 238 reflections of F > 0{sigma}(F) between 10 and 2.1 Å resolution.


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Table II. Data collection and refinement statistics for SB3 and SB4 mutants
 
Overall structure of SB3 in the asymmetric unit

The overall structures of SB3 and SB4 were almost the same, except around Lys462 and a few other residues. In both cases, the crystals have four molecules per asymmetric unit, while the wild-type SBA crystals contain only one molecule. Figure 3 illustrates the overall structure of SB3 in the asymmetric unit. In order to understand the intersubunit interactions better, subunit names were assigned arbitrarily to each of the four molecules in the asymmetric unit. The mutated residues were indicated by their respective colors. The interactions between molecule A–molecule D and molecule C–molecule B were almost the same and reciprocal, although they are not exactly symmetrical. The main interactions between molecule A and molecule D (also between molecule C and molecule B) were found in the region from Asn24 to Asp31 of one molecule and in that from Lys462 to Pro463 of the other molecule. A part of a flexible loop involved in the enzyme activity mechanism (residues 99–103) was also involved in the crystal interface interactions between molecule A and molecule D (as well as between molecule C and molecule B) with the residues from Arg162 to Glu170, Lys283 and Leu286.



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Fig. 3. Overall structure of the SB3 in an asymmetric unit. Mutated residues are indicated by different colors: red, D374Y; blue, L481R; green, P487D; magenta, K462S (only in SB4). Molecule A and molecule D, and molecule C and molecule B had almost the same interactions, making them reciprocal, although they are not exactly symmetrical.

 
The four molecules in the asymmetric unit were almost identical as reflected in the all atom r.m.s.d., which has ~0.02–0.20 Å between its four subunits. The r.m.s.d. of the main chain between wild-type SBA and the SB3 mutants at each subunit was revealed to be ~0.46 Å. The most significant side chain variations occurred at the surfaces, especially at crystal contact sites where interaction with neighboring molecules took place. This side chain rearrangement induced new crystal packing through the formation of new crystal contacts.

Interface interactions

Each of the four molecules in an asymmetric unit has a unique interface with individual molecules. However, the interactions between molecule A and molecule D are the same as the interactions between molecule C and molecule B, reciprocally. These crystal interfaces are not found in the wild-type SBA with its symmetric arrangement of the four molecules. Therefore, it was presumed that the formation of the new interfaces stemmed from the amino acid substitutions at the surface regions and the resulting change of crystal packing.

Figure 4 illustrates the interactions of crystal contacts between molecule A and molecule B. This interface can be largely divided into two sections. The first section of the interfaces is from Asp33 to Leu40 in {alpha}1 of molecule A and the L'7 (Gln401–Asn406) of molecule B. The second section is from Gln80 to Gly83 in L'2 of molecule A and Leu486 to Asp490 in the C-terminal loop of molecule B. Of special note is Asp487 in molecule B, which is the residue that was mutated from a proline. Around that residue, there were many intersubunit interactions including van der Waals contacts and hydrogen bonds. The hydrogen bonds at the interfaces in SB3 and SB4 are summarized in Table III. We concluded that the mutation of P487D is primarily responsible for the change of crystal packing.



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Fig. 4. Interface between molecule A and molecule B of the SB3 mutant. The interface can be separated largely into two sections. Hydrogen bonds are indicated by blue dashed lines and van der Waals contacts are in black. (a) Interactions are created between Gln80 and Gly83 of molecule A, and Leu486 and Asp490 of molecule B. The Asp487 mutated from Pro is indicated in red. (b) Another interface segment from {alpha}1 (Asp33–Arg43) of molecule A and L'7 (Gln401–Asn406) of molecule B.

 

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Table III. Hydrogen bonds between the interfaces in SB3 and SB4, respectively
 
SB3 and SB4 have very similar overall structures and interfaces. The two mutants differ at one position 462 that has lysine in SB3 and serine in SB4. However, there are a few differences in the crystal interfaces between these two mutants. Roughly speaking, SB3 has more interactions in a wider area of crystal interfaces than does SB4 (Figure 5). In SB3, there are some extra interactions between Lys462 of molecule B(D) and Asp26 of molecule C(A), and Ser464 of molecule B(D) and Glu30 of molecule C(A), which SB4 does not have. Furthermore, the positively charged nitrogen atom of Lys462 in SB3 makes a charged hydrogen bond with the negative oxygen atom of Asp26, while SB4 did not have any contacts around Ser462 (Table III). These charged hydrogen bonds were reciprocal between a pair of molecule A–molecule D and molecule C–molecule B, and could make the crystal tetramer of SB3 more compact than that of SB4.



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Fig. 5. Comparisons of the interface interactions between SB3 and SB4. SB3 has more interactions in a wide range of crystal interfaces than does SB4. The black dashed line indicates van der Waals contacts. (a) Interactions around Lys462 in SB3. There are extra interactions between Lys462 of molecule B(D) and Asp26 of molecule C(A), and Ser464 of molecule B(D) and Glu30 of molecule C(A). A charged hydrogen bond, indicated by a red dashed line, was formed between the positive nitrogen atom of Lys462 and the negative oxygen atom of Asp26. (b) Interactions around Ser462 in SB4. SB4 did not have any contacts around Ser462.

 
As one can see from Figure 6, the comparison of the {Delta}ASA under the tetramer formation (regarding the case of SB3 and SB4 as a kind of ‘tetramerization’ within an asymmetric unit of the crystal) also shows that there are more interface interactions in SB3 than SB4. It makes sense that SPB (the naturally occurring tetramer) has the highest value among these models. Excluding SPB, the order of increasing {Delta}ASA per subunit on the complexation is tSBA, SB4 and then SB3.



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Fig. 6. {Delta}ASA upon the tetramer formation of SPB, tSBA and two mutants. Excluding SPB, the order of increasing {Delta}ASA per subunit on the complexation is tSBA, SB4 and SB3.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The primary goal of this work was to create a stable SBA tetramer in solution based on the SPB tetramer structure. It is commonly accepted that the ASA at a certain residue is remarkably decreased when it makes an intermolecular interaction creating the buried surface dominated by the hydrophobic interaction. Therefore, as the first step of the design, we checked the changes in ASA at an interface that was formed when tetramerization took place, and some mutation sites were selected in the light of those changes. However, the overall goal to obtain a ‘stable tetramer in solution state’ was not achieved. Instead, we obtained new types of crystals that completely differed from native SBA crystals.

Some critical amino acid residues of SBA located at the active site were mutated for examining the enzyme activity mechanism [A.Hirata, M.Adachi, A.Sekine, Y.N.Kang, S.Utsumi and B.Mikami (2003) J. Biol. Chem., submitted for publication]. However, the crystal system and space group as well as the crystal form were nearly the same in the native and the mutated ones, being trigonal P3121. Contrary to this, the new type of variants mutated at the surface region produced a completely different crystal from the native. In this case, the crystal system was triclinic and the space group was P1, indicating that the crystal packing of the mutants was changed drastically compared with the wild-type SBA. We conclude that the surface mutation affected the surface-charge distribution significantly and in this way caused the change in the crystal packing.

In SB3, Asp374 was mutated to Tyr, Leu481 to Arg and Pro487 to Asp. Two hydrophobic residues in the C-terminal long loop were changed to the charged side chains. These substituted charged residues in the surface region might be involved in the interactions with the solvent and other molecules in a very new way. In a crystal, each protein molecule makes several contacts with its neighboring molecules. In fact, completely new interactions were observed in SB3 and SB4 that had never been found in the wild-type SBA crystals.

The mutations of SBA in this study did not affect the main chain structure significantly, but they did induce some side chain reorganization. Furthermore, a comparison of side chain conformations in SB3 with native SBA reveals that there are large differences between them, especially at the residues involved in intermolecular interactions. In other words, mutations on the surface region changed the intermolecular contact pattern within a crystal and this produced new crystal-packing interfaces.

From a practical point of view, two important findings can be gleaned from this research. First, if it is possible to control the quaternary structure by substitution or insertion/deletion at the key residues of the natural monomeric proteins, this will be very practical and applicable in the field of protein structure design. Recently, some studies about changing the quaternary structure—either monomer to oligomer (Kuhlman et al., 2001Go; O’Neill et al., 2001Go) or oligomer to monomer (Thoma et al., 2000Go)—by only a few key mutations, based on a structural and computational design procedure, have been reported. Ultimately, this can be used for regulating the protein functions or investigating the cause of the difference in quaternary structure of two proteins belonging to the same species, as well as the evolutionary relations. This is encouraging, because it does suggest a few ways to possibly redesign the SBA tetramer. As mentioned previously, both the SB3 and SB4 mutants from this trial have four molecules in an asymmetric unit, while there is one molecule in the equivalent unit of the native SBA crystal. This can in a way be interpreted as a ‘crystal tetramer’. If the interactions between four molecules in the asymmetric unit get stronger and more orderly, it can be a permanent tetramer in solution.

Secondly, to make crystals more stable by opportune protein engineering for better crystallographic resolution is a possible outcome. In fact, there exist examples of crystals in which the protein–protein contacts were engineered in order to improve the crystal quality (Lawson et al., 1991Go; Janin and Rodier, 1995Go). Learning which parts of the protein surface are involved in crystal-packing contacts will be very valuable (Carugo and Argos, 1997Go). The results presented in this paper indicate that mutation at only three amino acid mutations on the surface region was sufficient to change crystal-packing contacts significantly.

Despite many efforts for the discovery of protein–protein interactions, it is still difficult to predict exactly the effect of amino acid substitutions on protein structures, and the design of the protein structure to fulfill a certain demand remains a difficult process. Understanding the complexity of protein–protein interactions and dealing with the general lack of predictable ultimate effects on the overall structure will probably require many trial and error tests. Crystal analysis is a sensitive and accurate tool for investigation of these intermolecular interactions.

Though we could completely change the crystal packing of SBA by introducing three mutations on the surface, our experiment demonstrated that the mutations are not sufficient to make a stable tetramer or dimer in solution. In the case of SPB, interface I (Figure 1a) involves not only the three residues mutated in SBA but also the N- and C-terminal regions (Figure 1b). In the structures of SBA, determined so far by X-ray crystal analysis, five or six residues of the N-terminal region are difficult to fix in the model owing to their high temperature factors, in contrast to the clearly visible residues in SPB (Cheong et al., 1995Go). The two Pro residues, Pro3 and Pro5 in SPB, seem to make the peptide rigid to allow the three N-terminal residues to interact with the peptide of Val376 to Asp378 (Cheong et al., 1995Go). In the C-terminal region, SPB is longer by five residues (SNPFD) than SBA, which actually makes a good interaction with the C-terminal loop (residues 478–481) of the adjacent molecule at interface I (Figure 1). These results suggest that the five different N-terminal residues play a key role, especially the two rigid Pro residues, and that the five-residue long extension at the C-terminal region in SPB stabilizes the dimer in interface I. We are now designing a new mutant of SBA to test this hypothesis.

The atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank, with accession Nos 1UKO (SB3) and 1UKP (SB4).


    Acknowledgements
 
We are very grateful to Professor S.W.Suh at Seoul National University for providing us with the SPB coordinates information. We thank Professor Hiroaki Kato and the staff at Kyoto University for their guidance and technical assistance on the dynamic light scattering measurements used in the experiments. This work was supported by a grant of the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science and Technology.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received June 9, 2003; revised September 11, 2003; accepted September 16, 2003.





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