Review of the Cellulose Synthase

Cellulose

Cellulose is linear polymer derived from D-glucose units, which condense through β(1-4)-glycosidic bonds. [1]

In the cell the multiple hydroxyl groups on the glucose from one chain form hydrogen bonds with oxygen atoms on the same or on a neighbor chain, holding the chains firmly together side-by-side and forming microfibrils with high tensile strength. Several different crystalline structures of cellulose are known, corresponding to the location of hydrogen bonds between and within strands. Many properties of cellulose depend on its chain length or degree of polymerization, the number of glucose units that make up one polymer molecule. Molecules with very small chain length resulting from the breakdown of cellulose are known as cellodextrins; in contrast to long-chain cellulose, cellodextrins are typically soluble in water and organic solvents. [1]

Bacterial cellulose differs from the one in plants. In general, microbial cellulose is more chemically pure, containing no hemicellulose or lignin, has a higher water holding capacity and hydrophilicity, greater tensile strength resulting from a larger amount of polymerization, ultrafine network architecture. Furthermore, bacterial cellulose can be produced on a variety of substrates and can be grown to virtually any shape due to the high moldability during formation. Additionally, bacterial cellulose has a more crystalline structure compared to plant cellulose and forms characteristic ribbon-like microfibrils. A hallmark of microbial cellulose, these thin microfibrils are significantly smaller than those in plant cellulose, making bacterial cellulose much more porous. [2]

Bacterial cellulose appears to fulfill diverse biological roles within the natural habitat, conferring mechanical, chemical, and physiological protection or facilitating cell adhesion during symbiotic or infectious interactions in different species. [3] Also cellulose mediates cell-cell interactions, cell adherence and biofilm formation on biotic and abiotic surfaces. [4]

Organism

The object of the review is received from Rhodobacter sphaeroides - purple bacterium capable for photosynthesis, anaerobic phototrophy and aerobic chemoheterotrophy in the absence of light. [5]

Complex

*Press "Start script 9" to see each ligand, please (shows all the ligands except 3PE).

Complex include two subunits of bacterial cellulose synthase- A and B, ligands - 3-SN-phosphatidylethanolamine, cyclic dimer of diguanosine monophosphate, diundecyl phosphatidyl choline, uridine-5'-diphosphate and magnesiun ion.

• BcsA
• PDB ID: 4P00
• Uniprot ID: Q3J125
• BcsA consists of eight transmembrane (TM) segments and two large cytoplasmic domains, a glycosyltransferase domain in the middle and a C-terminal fragment that contains the c-di-GMP-binding PilZ domain. [4]

• BcsB
• PDB ID: 4P00
• Uniprot ID: Q3J126
• BcsB is located in the periplasm and is anchored in the membrane by a single transmembrane helix. [4] BcsB Is required for catalytic activity of BcsA.[7]

BcsA-B complex reveals a complete cellulose biosynthesis cycle, from substrate binding to polymer translocation. [6] Bacterial cellulose synthase polymerizes glucose molecules via β-1,4 glycosidic linkages in a multi-step process which requires the presence of a divalent cation, mostly magnesium. First, upon stimulation by c-di-GMP, the enzyme binds its substrate uridine diphosphate(UDP)-activated glucose (donor) at an intracellular glycosyltransferase domain. Second, the donor glucose is transferred to the 4′ hydroxyl group at the non-reducing end of the growing polysaccharide chain (acceptor), thereby extending the polymer and forming UDP as a second reaction product. Third, following glycosyl transfer, the elongated polymer has to be translocated by one glucose unit into a transmembrane (TM) channel so that the newly added glucose unit occupies the acceptor site and UDP must be replaced with UDP-Glc for another round of catalysis. [7]

Cellulose is synthesized by membrane-integrated glycosyltransferases that contain 6 to 8 transmembrane helices as well as an intracellular catalytic glycosyltransferase domain. These enzymes polymerize UDP-activated glucose (UDP-Glc) into chains thousands of glucose units long and translocate the polymer across the plasma membrane, through a pore formed by their own TM region. [6], [9]

Processive (processivity is an enzyme's ability to catalyze consecutive reactions without releasing its substrate) GTs, including chitin, alginate and cellulose synthases, transfer the glycosyl moiety from a nucleotide-activated sugar (donor) to a specific hydroxyl group of the growing polysaccharide chain (acceptor) by a nucleophilic SN2 (type of nucleophilic substitution reactions, where one bond is broken and one bond is formed synchronously, in one step)-like substitution reaction, thereby forming an elongated polymer and nucleoside diphosphate as reaction products. A processive mechanism requires that the elongated polymer is translocated after each glycosyl transfer, such that the polymer’s newly added sugar unit becomes the acceptor in a subsequent reaction. Because all known processive GTs are TM channel-forming enzymes, the translocation of the polymer into the TM channel between catalytic steps also gives rise to secretion. [6]

BcsA translocates cellulose via a ratcheting mechanism involving a short helix within the GT domain - “finger helix” - that contacts the polymer's terminal glucose with hydrogen bonds. Cooperating with BcsA's gating loop, the finger helix moves ‘up’ and ‘down’ in response to substrate binding and polymer elongation, respectively, thereby pushing the elongated polymer into BcsA’s transmembrane channel. All states of BcsA observed thus far either show its gating loop inserted into the active site and the finger helix in the ‘up’ position (pre-translocation state) or the gating loop retracted from the active site and the finger helix in the ‘down’ position, if the cellulose polymer is extended. [6]

Ligand cyclic dimer of diguanosine monophosphate (c-di-GMP):

• IUPAC: 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)- 3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro- 2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine- 2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin- 6-one)
• Empirical formula: C20 H24 N10 O14 P2
• Molar mass: 690 g/mol

Signaling molecule cyclic-di-GMP (c-di-GMP), a potent biofilm inducer and allosteric (allosteric regulation is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme's active site) activator of BcsA. In the biosynthesis it binds with a β-barrel of BcsA via hytrogen bonds, salt bridge and π–π stacking interactions. [7]

Ligand uridine-diphosphate (UDP):

• IUPAC: Uridine 5'-(trihydrogen diphosphate)
• Empirical formula: C9 H14 N2 O12 P2
• Molar mass: 404 g/mol

It is an ester of pyrophosphoric acid with the nucleoside uridine. UDP consists of the pyrophosphate group, the pentose sugar ribose, and the nucleobase uracil. [8] It takes part of elongation of the cellulose in assembly with uridinediphosphate-activeted molecule and then releases as a result of connection glucose and polymer. [6]

UDP is involved in the elongation of cellulose, presents as a part of UDP-Glc before elongation and releases after glucose molecule joins the polymer. [6]

Ligand β-D-glucose:

• IUPAC: (2R,3R,4S,5S,6R)-6-(hydroxymethyl)oxane-2,3,4,5-tetrol
• Empirical formula: C6 H12 O6
• Molar mass: 180 g/mol

Monomer of cellulose.

Ligand magnesiun ion (Mg2+):

• As UDP is involved in polymer elongation.

Materials and methods

• JMol, JSMol
• CluD
• Method of studying the object:
  1. Method of studying the srtucture:
     • We studied the structure of the protein and ligands by defferent painting in JMol. We colored them by: chains, structure, hydrophobic cores. And we used different models: cartoon, cpk, backbone.
     • We found out the active site of the protein by selecting those parts which were at distance less than 6Å from the ligands.
  2. Method of studying the contacts (we selected ligands and then...):
     • We selected backbone to see the covalent bones and used measurements to show the length of the bond.
     • We selected the hydrogen bones (as inside the protein as inside the ligands as between ligands and protein) and used measurements to show the length and the angle of the bond.
     • We selected carged aminoacids: ASP, GLU, ARG, LYS, HIS - and then we selected those which were at distance less than 4Å - to find out the salt briges.
     • We used CluD to found out the hydrophobic cores and selected them and selected the rest of the protein to show surface of the protein and hydrophobic contacts with ligands.
     • To represent large region of secondary structure (β-barrel in the subunit A and membrane part of the protein complex, consisting of α-helix) we use the command (within (group, …)). We selected a few centers – few amino acids in the center of region – and suitable radius so, that all necessary elements of secondary structure were distinguished. Element of proteins, which have no attitude to considered structures were deleted manually.

Results and Discussion

* We are unable to do something with 3PE, so here is review only these ligands: UDP, PLC, MG, BGC, C2E.

Here is the applet with illustrations. The first script shows common characteristics of the protein: chains, secondary structure, Hydrophobic cores, surface, ligands. The second script shows different view options of active site of the protein and ligands. The third script shows contacts between protein and ligands.

* Scripts 1, 3, 7 take a long time for load.

If you want to see different representation of the protein, press "Start script" and then "Resume", please. If you want to save picture press "Save screen shot", please.

Table 1. Contacts between ligands and protein. Contact Number Covalent bonds 0 Hydrogen bonds UDP 9 PLC 2 BGC 15 C2E 13 MG 0 Salt bridges UDP 9 PLC 2 BGC ? C2E 8 MG 4 Hydrophobic interactions UDP 2 PLC 2 BGC 0 MG 0 C2E 2

Table 2. Contacts inside the protein. Contact Number Covalent bonds all over the backbone Hydrogen bonds 809 Salt bridges 71 Hydrophobic cores one huge and one big cores Disulfid bonds 1

Abstract of results:

• There are two chains in this protein. (You can see it by starting and resuming the script 1)
• There are ligands: UDP, PLC, MG, BGC, C2E, (and 3PE does not demonstrated). (You can see it by starting and resuming the script 3)
• There is hydrophobic area on the surface in the middle part of the protein assembly. (Start script 7 and resume to see it) This part of the protein is builded with α-helixes. There is the structure builded by β-sheets which called β-barrel.
  There is only one disulfid bonde in the protein (:B). (Start script 8 and resume to see it)
• There are hydrophobic interactions between protein and ligands except MG and BGC. (Start script 10 and resume to see it) (see table 1)
• The ligands are surrounded by aromatic residues (starting with 4Å). (You can see it by starting and resuming the script 2)
• There are four TRP among that residues. (You can see it starting the script 2)
• The protein and the ligand do not connect by covalent bonds. But backbone atoms of the ligands as well as backbone atoms of protein are connected with covalent bonds. The typical length is 1.5Å. (Start script 4 and resume to see it) (see table 1)
• The protein and the ligands, except MG, connect by 39 hydrogen bonds. The average length of the displayed bonds is 2,94 Å and the angle is 133.3 °.(Start script 5 and resume to see it) (see table 1)
• There are 155 salt bridges between protein's atoms. And there are not salt bridges between protein and ligands. The typical length of the salt bridge is 2.5Å. (Start script 6 and resume to see it) (see table 1)

Interactions inside cellulose synthase complex

Interaction with cellulose. The fragment of the cellulose (from pdb) located in a fairly large hydrophobic deepening between α-helixes in the membrane part of the protein assembly. These helixes are belonging to the subunits A and B (see script 7). There are a localisation of aromatic residues including four tryptophans - fairly rare type of hydrophobic amino acids. Also there are many hydrogen bonds connecting cellulose with charged groups of the protein chain. These bonds ensure smooth promotion of the growing cellulose chain through the membrane.

Subunits' interaction. The interaction of the two subunits is located near the outer surface of the membrane. It is the transmembrane assembly of the α-helixes in subunit A which connect with perpendicular α-helixes in subunit B. There is the transmembrane terminal α-helix in subunit B. It is curved at an angle more than 90° and it lyes parallel for α-helixes in subunit A.
Subunits' connection is provided at least by 3 salt bridges, many hydrogen bonds, hydrophobic interactions; there are not any disulfide bond between subunits.

Ligands and subunit AAs we said in the review: subunit A is the allosteric enzyme. There are 3 different allosteric regulators (effectors) connected to this subunit as well as magnesium ion and a molecule of uridine-5’-diphosphate (UDP) located near the active site and involved in the elongation of cellulose synthesis.
We have studied the contacts between different types of ligands and protein. Allosteric regulator c-di-GMP (cyclic di-guanosine monophosphate) is a rather hydrophilic molecule. It binds to a subunit A right under the β-barrel, which is located on the periphery of the protein and most removed from the membrane (so it "deeper" immersed in the cytoplasm, where c-di-GMP locates).
On the contrary, ligands with point charges and long hydrophobic tails (3-phosphatidylethanolamine and DIUNDECYL PHOSPHATIDYL CHOLINE) interact with the protein in the transmembrane region, among hydrophobic amino acid residues.
The ligands located active site of the protein (them are required for catalytic activity) is extremely hydrophilic, is surrounded by protein and are so close to each other that they form a "mutual" salt bridge.

Intramolecular and intermolecular interactions

Hydrophobic interaction. Hydrophobic properties of the complex. There are more than 50 hydrophobic cores discovered totaly. But only 2 cores (274 and 2227 atoms) was quite large, the others were less (up to 20 atoms). As we expected, the most extensive hydrophobic areas on the surface of the protein assembly located in the transmembrane part. This part of the protein is builded by α-helixes with many hydrophobic residues. To sum up, it forms a hydrophobic "shell" (facing the membrane) and hydrophobic inner "channel", which was described in the previous paragraphs. This channel is used for moving the growing polysaccharide chain during the synthesis process.
Hydrophobic interactions are also involved in maintaining contact between the two subunits of the complex - there is a few small clusters of hydrophobic side chains of amino acids in this area. The interaction between ligands and subunit A, discussed in this work, is possible also due to hydrophobic interactions (see table and section "Interaction with cellulose")

Disulfide bonds. There is only one disulfide bridge (subunit A) around the protein assembly. And there are 7 CYS belonging subunit B which do not connect by S-S bridge. We assume that such a small number of disulfide bridges due to the position of the complex: both subunits are anchored inside the membrane and they are connectet together. In addition subunit B are surrounded by extracellular matrix. So because of mechanical interaction of the protein chains with water molecules is negligible - additional reinforcement structure by using S-S bridges is not required.

Hydrogen bonds. One of the most important role of hydrogen bonds in any protein is a formation and stabilization of secondary structure (see paragraph “Secondary structure”). Besides that, hydrogen bonds take part in organization of tertiary structure in both protein chains (see script 5) and in formation of quaternary structure of our complex (see script 8). Also we represent hydrogen bonds between protein complex and ligands (only ion of Mg does not form hydrogen bonds) – for more information see the table 1 and paragraph “Interactions inside cellulose synthase”.

Salt bridges.Salt bridges stabilize a tertiary structure and also provide a bond between 2 protein subunits (we found 3 salt bridges in area of contact of A and B subunits) and between proteins and ligands (see table 1) – localization of all salt bridges you can see in script 6.

Organization of protein chains

Secondary structure. There are different types of secondary structure in our protein: you can see a α-helixes, β-sheets (parallel and antiparallel β-sheets) and also reversive turns. One of the α-helix of the subunite, mentioned in a few articles as finger-helix, located near the active site of the protein. It takes part in synthesis and translocation of the growing cellulose.
*Representation of different secondary structure you can see in scripts.

Tertiary structure. Subunits A and B in our complex are quite different in their structure. Membrane part of cellulose synthase consists of asymmetrical “barrel” of α-helix, which form a hydrophobic surface, turned to membrane, and also hydrophobic canal in center (for more information see paragraph “Hydrophobic interactions”). In the subunit A we have also found β-barrel, binding with one of the ligand - allosteric regulator - the cyclic dimer of diguanosine monophosphate. β-structures, which located in subunit B are 2 regions, consisting of 2 layers of β-sheets – this structures are quite similar with squashed β-barrel.

Quaternary structure. Different chemical interactions between 2 protein subunits of cellulose synthase were discussed in detail in paragraph “Interection between subunit”. Growing cellulose binds with as A subunit as B subunit.

Conclusion

• It requires large hydrophobic semi-cylindrical channel which is placed the growing polymer for cellulose synthesis and transport across the membrane.
• Subunits' connection is provided by salt bridges, many hydrogen bonds and hydrophobic interactions.
• The protein activity is provided by a complex structure such as cylindrical form structure builded by α-helixes and the β-barrel. In addition, the presence of ligands UDP, PLC, MG and C2E is necessary for the operations.

Supplementary materials

Texts of the scripts (pr3, block 1, term 2):

* because of features of JSMol we had to paste "hbonds off" at the top of every script, so now you can run them in any order.
** because of features of JSMol we had to delete every measurements at the end of every script (the last step), so now you can run them again many times and in any order.

Acknowledgments

Author contributions: SI chose an object of study. SE wrote the literature review (RU and EN). SI studied ligands and secondary structure of the protein. PD wrote scripts number 1, 2, 3, 4, 7, 9 and part of scripts 5 and 8 (common characteristics of protein and ligands, active site, covalent bonds, hydrogen b., hydrophobic cores, ligands). SE wrote first part of script 6 (salt briges), and part of the script 5, SI wrote script 10, the first part of script 8 (secondary structure) and second part of script 6. The english version of website was imposed by PD. The russian version of website was imposed by SE (and SI). Text of the Results and Discussion was written by SI with help of PD and SE.

References