## 4 Results I: MIP and LacI families
### The LacI family  
As a second test of the usefulness of SDPhound, we apply it to investigating the LacI family, another classic testing ground for these methods. The LacI family is a rather large family of bacterial transcription factors, which comprises 54 orthologous and paralogous proteins divided in 15 subfamilies (AraR, KdgR, CcpA, DegA, YjmH, RbsR, PurR, CytR, GalSR, AscG, LacI, TreT, GntR, IdnR and FruR), populated with a number of members which varies from 2 (for the AraR, KdgR, YjmH, LacI and IdnR subfamilies) to 12 (for the CcpA subfamily). Further details on the nature of the set can be found in the references mentioned above, while here we summarize only the minimal data required for benchmarking SDPhound. The sequence alignment was the same as in [2; 19; 20]. Also for this family, key positions for discriminating among the mentioned subfamilies are searched for. And also in this case, the evaluation of positions indicated by [PERSON] and coworkers in [2] is used to compare the performances of our method and of SDPpred. In that work, SDPs were mapped on 3D X-ray structures 1 WET, 1 JWL and 1 BYK and classified into 4 groups, that we indicate with the numbering 1 to 4 in the score columns, marked as S, of Table 3. The classification is as follows: 1) experimentally validated SDPs, 2) SDPs of a domain, with a distance \(<5\)A from the effector, the substrate or another subunit within a multimer in at least one of the 3D structures and \(<7\)A in the other two, 3) SDPs in the vicinity of one of the domains, and 4) SDPs with no apparent function. As it can be seen from the Table, this test case too shows equivalence of the new and of the benchmark method. The performances of SDPhound with the identity substitution matrix and of SDPpred are practically equivalent, with close numbers of positions in the four classes indicated above (5 vs. 4 in class 1, 12 vs. 13 in 2, 17 in 3 and only 1 in 4 in SPDhound vs. SDPpred). Positions 73, 190 and 292 are part of the binding site and interact very closely with the substrate. [PERSON] and coworkers [24] have investigated position 190, demonstrating its role in corepressor binding. This position was not predicted by SDPpred but was identified both by the B45 and the Bloc SDPhound runs. When analyzed with the pigeonholing runs, several interesting features of the relevant positions emerge, as summarized in Table 4. The experimentally validated positions (15, 16, 55, and 147) rank quite high in all physical characterization, pointing to the high specificity of the residues at those locations. Position 55 hosts a residue in close contact with operator DNA. The positive charge of K55 in 1 WET, as well as other polar residues able to donate a hydrogen bond found in the same position in other ortho/paralogous proteins, explains why it has been found by mutagenesis as a position influencing affinity for the operator DNA [25]. [PERSON] and coworkers demostrated that the positively charged Lys55 is able to discriminate among different operator DNA sequences. Firstly, it increases affinity for the electrostatically negative minor groove of the operator DNA; secondly, it selectively binds operator DNA's that does not contain guanine in the position Lys55 interacts with [26]. Nonpolar residues, such as alanine, appear in other paralogous proteins that can bind guanine-containing operator DNA sequences. SDPhound not only predicts this position, but it correctly points out that the physical property determining its specificity is based on its charge/polarity. We would like to emphasize that such result is obtained by SPDhound without any structural information of the protein or the interacting DNA. Among the other positions in Table 4, 98 stands out both for charge and size relevance. Inspection of the X-ray structures reveals that residue 98 (or its homologous 100 in PDB structure 1 JWL) is located both at the opening of the substrate pocket and in contact with another protein in the dimer.