Alpha-enolase (ENO1) is a multifunctional protein with oncogenic roles. First described as a glycolytic enzyme, the protein performs different functions according to its cellular localization, post-translational modifications, and binding partners. Cell surface-localized ENO1 serves as a plasminogen-binding receptor, and it has been detected in several cell types, including various tumor cells. The plasminogen system plays a crucial role in pathological events, such as tumor cell invasion and metastasis. We have previously demonstrated that the interaction of ENO1 with the multifunctional chaperone Hsp70 increases its surface localization and the migratory and invasive capacity of breast cancer cells, thus representing a novel potential target to counteract the metastatic potential of tumors. Here, we have used computational approaches to map the putative binding region of ENO1 to Hsp70 and predict the key anchoring amino acids, also called hot spots.