Factors Governing the Different Functions of Zn2+-Sites with Identical

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Factors Governing the Different Functions of Zn2+-Sites with Identical Ligands in Proteins Yu-Ming Lee, Cedric Grauffel, Ting Chen, Karen Sargsyan, and Carmay Lim J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.9b00617 • Publication Date (Web): 30 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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Journal of Chemical Information and Modeling

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Factors Governing the Different Functions of Zn2+-Sites with Identical Ligands in Proteins ††

††

††

Yu-Ming Lee‡*, Cédric Grauffel‡, , Ting Chen‡, , Karen Sargsyan‡, , and Carmay Lim‡§* ‡Institute §Department

of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan of Chemistry, National Tsing Hua University, Hsinchu 300 Taiwan

Abstract In Zn-proteins, structural Znsites are mostly Cys-rich lined by two or more Cys residues, whereas catalytic Zn-sites usually contain His or Asp/Glu residues and a water molecule. Here, we reveal many examples outside this trend with Zn2+ bound to ligands commonly found in both structural and catalytic Znsites, namely, Zn-CC(C/H)x (x = D, E, or H2O) sites. We show that these atypical Zn-sites are found in all known life forms (i.e., eukaryotes, bacteria, archaea, and viruses) and can serve structural roles in some proteins but catalytic roles in others. By calculating the physical properties of these atypical Zn-binding sites, we elucidate why Zn-CC(C/H)x sites of the same composition can serve structural and catalytic roles in proteins. Furthermore, we found new sequence/structural motifs characteristic of catalytic Zn-CCHw sites and provide guidelines to predict the structural/catalytic role of atypical Zn-CC(C/H)x sites of unknown function. We discuss how our results could help to design inhibitors targeting catalytic Zn-CC(C/H) H2O sites.

Keywords: Zinc, catalytic Zn, structural Zn, Zn enzymes, bond-valence method, metal ions

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Introduction Zn2+ is an essential cofactor in numerous enzymes and regulatory proteins.1-9 Many Zn-proteins are therapeutic drug targets such as metallo--lactamase,10 breast cancer type 2 susceptibility protein (BRCA2),11 carbonic anhydrase,2,

12-14

carboxypeptidase A,3,

15, 16

histone deactylase,17

matrix

metalloproteinase,18 and nucleocapsid protein 7.19 In proteins, most Zn-binding sites are either (1) “structural” where Zn2+ serves solely to stabilize the protein structure and/or (2) “catalytic” in which Zn2+ participates in an enzyme-catalyzed reaction by (a) polarizing/activating a water molecule, (b) facilitating substrate binding, or (c) stabilizing a transition state/intermediate directly. These two types of Zn-binding sites generally exhibit different solvent accessibility and ligand preferences.20-23 Structural Zn-sites such as Zn-CCCC, Zn-CCCH, and Zn-CCHH found in Zn-finger proteins are mostly buried and contain two or more Cys ligands. (Note that the notation Zn-xxxx denotes Zn2+ tetra-coordinated to four residues that are all not contiguous in the primary sequence.) In contrast, catalytic Zn-sites such as Zn-HHHw (where w denotes a water ligand) in carbonic anhydrase and ZnHHH(D/E) in carboxypeptidase A are more solvent-exposed and usually contain Asp/Glu and neutral His or water ligands. We had provided a physical basis for the observed preference for cysteines over neutral histidines in structural Zn-sites on the basis of three physico-chemical features: (1) the extent of charge transfer from the ligands to the metal cation, which depends on the distance from the metal-ligating atom to the cation, (2) the size of the Zn2+-ligating atom, and (3) the solvent accessibility of the Zn-site.9, 22 Compared to other amino acid (aa)/water ligands, Cys− transfers more charge to Zn2+, thereby reducing the metal’s positive charge and electron-accepting ability. This along with the size of the bulky Cys (S−) compared to that of smaller O/N metal-ligating atoms prohibit Zn2+ in buried CCCC, CCCH, and CCHH sites to bind another ligand. On the other hand, non-buried Zn-sites containing only one or no Cys− ligands enable Zn2+ to be a good electron acceptor and to serve a catalytic role. Hence, a simple way to distinguish structural Zn-sites from catalytic ones is to simply count the number of Zn-bound Cys− and water ligands: When Zn2+ is tetrahedrally bound to only aa ligands comprising two or more Cys−, it is likely to play only a structural role, but when it is coordinated to 1 Cys− and a water molecule, it can play a catalytic role. These simple criteria yielded a ~95% accuracy in predicting the role of Zn2+ in 109 nonredundant Zn-sites (45 structural and 65 catalytic) in 79 protein families.9, 22 2 ACS Paragon Plus Environment

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3 However, interactions beyond the first-shell ligands; i.e., the protein matrix, can also influence the role of Zn2+. The protein matrix can modulate the pKa of the Zn-bound water molecule (denoted as pKw) by (i) changing the metal coordination number, (ii) providing hydrogen-bonding interactions with second-shell residues and/or electrostatic interactions with nearby charged aa residues, and (iii) controlling solvent access to the Zn-site.23, 24 Hence, for the same set of ligands, Zn2+ can play a structural role in one protein but a catalytic role in another. Indeed, we noticed that a few Zn-CC(C/H)x (x = D/E or H2O) sites, resembling Zn-finger cores, serve structural and catalytic roles according to the literature. For example, the Zn-CCHE site in the C-terminal RNA-dependent RNA polymerase of the Lassa virus conserved nucleoprotein (Figures 1a, 1b) plays a structural role,25 whereas that in cysteinyl-tRNA synthetase (CysRS) (Figure 1c, 1d) plays a catalytic role.26

Figure 1. Example of a dual-role Zn-site. The Lassa virus nucleoprotein (PDB 3r3l) (a) has a structural Zn-CCHE site (b). The cysteinyl-tRNA synthetase (PDB 1li5) (c) has a catalytic Zn-CCHE site (d). 3 ACS Paragon Plus Environment


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4 Our observations lead us to ask the following intriguing, fundamental questions: 1. Do atypical Zn-CC(C/H)x (x = D, E or H2O) sites resembling Zn-finger cores but with an Asp/Glu/H2O ligand commonly found in catalytic Zn-sites occur in all known life forms or are they found only in specific species? 2. Do all Zn-CC(C/H)x sites serve dual structural/catalytic roles? 3. What factors dictate the structural/catalytic role of a Zn-CC(C/H)x site in a given protein? To address these questions, we screened the entire Protein Data Bank (PDB)27 to find all Zn-CC(C/H)x sites (x = D, E or H2O). These were then grouped according to the four domains of life; viz., eukaryote, bacteria, archaea, and viruses. For each Zn-CC(C/H)x site found, we combed the literature to determine if it is reported to play a structural/catalytic role. To elucidate why a Zn-CC(C/H)x site plays a structural role in one protein but a catalytic role in another, we assessed the effects of the Cys− ligands and second-shell residues on the Zn-pKw as well as the protein matrix in modulating solvent entry into the Zn-site and altering the Zn2+ligand distances. Based on our findings, we provide guidelines to predict the structural/catalytic role of a Zn-CC(C/H)(D/E) site in a given protein, and used them to predict the role of Zn-sites whose functions are unknown in proteins.


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Results Zn-CC(C/H)x sites are ubiquitous in proteins. To determine if Zn-CC(C/H)x sites (x = D, E, or w) occur in all known life forms, we screened the PDB27 (2019, February) containing 148,886 entries for structures containing Zn-CC(C/H)x (x = D, E, or H2O) sites in eukaryotic, bacterial, archaeal, and viral proteins. Multinuclear Zn-CC(C/H)x sites were excluded by checking that no other metal ion is within 5 Å of Zn2+ in each structure since the metalmetal distances in polynuclear sites generally do not exceed 5 Å.28 First, we checked the identity of Zn2+ and its ligands using the metal-binding site validation server, CheckMyMetal (http://csgid.org/csgid/metal_sites),29,

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which employs eight

experimental parameters (occupancy, B-factor, ligands, valence, nVECSUM, geometry, gRMSD and vacancy) to assess the quality of each modeled metal-binding site. Only mononuclear Zn-CC(C/H)x sites with full occupancy of Zn2+ (occupancy = 1) and all coordination sites corresponding to a given geometry (vacancy = 0) were included. This resulted in 73 Zn-CCCD, 46 Zn-CCCE, 167 Zn-CCCw, 86 Zn-CCHD, 162 Zn-CCHE, and 216 Zn-CCHw sites. For each type of life form, we clustered protein sequences containing atypical Zn-sites sharing 30% sequence identity using the CD-HIT program (http://weizhongli-lab.org/cd-hit/31 and chose the protein with the highest-resolution structure as the cluster’s representative. This yielded 30 eukaryotic, 31 bacterial, 7 archaeal, and 6 viral nonredundant proteins (Supplementary Table S1), indicating that Zn-CC(C/H)x sites occur in all life forms. Zn-CC(C/H)x sites can play structural/catalytic roles in proteins. For each representative protein, we assigned the structural/catalytic role of the Zn-CC(C/H)x site according to the literature. A Zn-CC(C/H)x site was assigned “catalytic” if it was reported to participate in the enzyme-catalyzed reaction; otherwise, it was assigned a structural role. To verify the functional role of

the

Zn-CC(C/H)x

site,

we

used

the

Conserved

Domain

Database

(CDD)

(https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)32 to identify a conserved domain, if any, containing the Zn-site and the role annotated by the CDD. The results in Figure 2 indicate that the 31 nonredundant bacterial proteins exhibit a higher ratio of catalytic (orange bars) to structural (blue bars) Zn-CC(C/H)x sites, as compared with the 30 nonredundant eukaryotic proteins.



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Figure 2. The percentage frequency distributions of nonredundant proteins with catalytic (orange) and structural (blue) atypical Zn-CC(C/H)x sites (x = D, E, or w) for each type of life form. All six atypical Zn-sites play structural roles in some proteins and catalytic roles in others. One factor governing the structural/catalytic role of the Zn2+ in these proteins that is apparent in Figure 3 is the number of Zn-bound cysteines in sites containing a water ligand. The ratio of catalytic to structural Zn-CCHw sites is far larger than that in Zn-CCCw sites, indicating that reducing the number of Zn-bound cysteines increased the likelihood of catalytic Zn-CCHw sites.

Figure 3. The number of nonredundant proteins where Zn2+ plays either a structural (blue) or a catalytic (orange) role for each of the atypical Zn-sites.


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7 Effect of 1st shell ligands on Zn-site reactivity. One way in which the number of cysteines in Zn-CC(H/C)w sites might affect the role of the Zn2+ is to alter the Zn2+-bound water protonation state.33 To evaluate how the number of Zn-bound Cys− affects the Zn2+-bound water protonation state, we computed the pKw values of Zn-HHHw, Zn-CHHw, Zn-CCHw and Zn-CCCw sites in various environments modeled by an effective dielectric constant  ranging from 10 to 30 to reflect increasing solvent exposure of the Zn-site (see Methods). Note that the protocol used to compute the pKw could reproduce the experimental pKw of 8 for the Zn-HHHw complex in water.24 Increasing the number of Zn-bound Cys− prohibits Zn2+-bound water deprotonation. Replacing neutral His in the Zn-HHHw site successively by negatively charged Cys− increased the pKw (Figure 4); e.g., the pKw values of non-buried Zn-HHHw, Zn-CHHw, Zn-CCHw, and Zn-CCCw sites characterized by  = 30 are 7.4, 8.1, 10.5, and 15.8, respectively. Increasing the number of anionic Cys− hinders deprotonation of the Zn-bound water molecule due to increased charge-charge repulsion with the hydroxide, resulting in lower acidity (increased pKw) of the Zn2+-bound water molecule.

Figure 4. The pKw values of Zn-sites as a function of the number of Zn-bound Cys: 0 Cys (ZnHHHw), 1 Cys (Zn-CHHw), 2 Cys (Zn-CCHw), and 3 Cys (Zn-CCCw). The filled triangles and squares correspond to pKw values computed for  = 30 and  = 10, respectively. Effect of 2nd shell. If Cys ligands generally disfavor deprotonation of the Zn-bound water molecule, how can Zn-CC(C/H)w sites play catalytic roles? Upon examining all the catalytic ZnCC(C/H)w sites in protein structures, we found that the Zn-bound water molecule is hydrogen-bonded to an Asp/Glu carboxylate in all the structures except in the cyclase enzyme, NisC (PDB 2g0d, 7 ACS Paragon Plus Environment


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8 Supplementary Table S2). The NisC enzyme does not require a 2nd-shell Asp/Glu to activate the Zn-bound water molecule, which is displaced by a substrate in the proposed reaction mechanism.34 We performed multiple structure alignment of nonredundant protein fragments containing these catalytic Zn-CC(C/H)w sites using mTM-align (http://yanglab.nankai.edu.cn/mTM-align/). Each fragment was obtained by cutting out a protein subsequence that starts with the first Zn-ligand in the sequence terminating with the last Zn- ligand, capped by the preceding and the following residue along the sequence at the N- and C-terminal end, respectively. The multiple structure alignment yielded two groups with >3 structures each, which belong to the same branch of the structure-based phylogenetic tree (Supplementary Table S3). These two groups yielded distinct locally conserved structures (Figure 5).35 Multiple sequence alignment for each group yielded the corresponding sequence motifs shown in Figure 5, where the Zn2+-ligands are underlined in bold and the second-shell D/E is in red. (a) (H/C)AEx2(A/V)x35PCxx(C/H)

(b) CxDxRx77HxxC

Figure 5. Sequence and structural motifs characterizing catalytic Zn-CC(C/H)w sites. The sequence motifs (left) comprise the Zn2+-ligands (bold underlined) and conserved residues including the secondshell D/E (red). The sequence logo was drawn using WebLogo (https://weblogo.berkeley.edu), where the height of each 1-letter aa code (measured in bits) reflects the relative frequency of the aa residue at that position; the larger the height, the higher the conservation of the aa residue at that position. The superimposed structures (right), shown in different colors and transparency levels, share

Factors Governing the Different Functions of Zn2+-Sites with Identical

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